Symposium S.SM01—Organ-on-a-Chip—Toward Personalized Precision Medicine

We will discuss the synthesis of carbon nanotubes and nanotube networks using different dopants during chemical vapor deposition. In particular, the effects of sulfur, boron and nitrogen will be discussed. It will be demonstrated that it is indeed possible to assemble/grow carbon nanotube networks if a careful control of dopants is achieved during chemical vapor deposition (CVD) growth. High-resolution electron energy loss spectroscopy (HR-EELS) studies on these nanotube materials will be presented, and the locations of boron, sulfur and nitrogen within nanotubes will also be shown. First principles theoretical calculations on nanotubes containing pentagon, hexagons and heptagons in the presence of these dopants will be discussed. We will also discuss the cytotoxicity and applications as molecular sensors and virus traps of these doped nanocarbons. This talk will also discuss the synthesis of large-area, high-quality monolayers of nitrogen-, silicon- and boron-doped graphene sheets on Cu foils using ambient-pressure chemical vapor deposition (AP-CVD). Scanning tunneling microscopy (STM) and spectroscopy (STS) reveal that the defects in the doped graphene samples arrange in different geometrical configurations exhibiting different electronic and magnetic properties. Interestingly, these doped layers could be used as efficient molecular sensors in conjunction with Raman spectroscopy. In addition, the synthesis of hybrid carbon materials consisting of interconnected graphene layers (graphene foams) by solvothermal routes will be discussed. These foams are highly conducting, very robust and can operate at temperatures ranging from 77K to 1173K.

References:
[1] R. Cruz-Silva, M. Terrones, et al. (2014) ACS Nano 8, 5959-5967
[2] Y. Wu, Terrones, M. et al. (2015). Nature Communications 6, 6141; doi:10.1038/ncomms7141.
[3] Y.-T. Yeh, M. Terrones, et al. (2016) Science Advances 2: e1601026.
[4] S. Feng, M. Terrones, et al. (2016). Science Advances 2, e1600322.
[5] S. Feng, M. Terrones, et al. (2017) Nanoscale Horizons 2, 72-80.
[6] Y.-T. Yeh, M. Terrones, et al. (2019) PNAS, under review.

Heart failure following myocardial infarction (MI) is the number one cause of death in the United States, and western world due to the myocardium’s inability to regenerate to a significant degree once fully matured, leaving behind a fibrous scar tissue that is unable to contract or propagate electrical impulses. Cardiac tissue engineering (TE) is a field of biomedical engineering that aims to develop materials for the treatment of patients suffering from heart failure after MI. Development of these materials may involve electrospinning, a technique utilized for engineering fibrous scaffolds for tissue engineering applications. Here, we introduce electrospun cardiopatches based on gelatin methacryloyl (GelMA), due to its tunable mechanical properties, favorable biodegradation, and the presence of cell binding sites, which promote adequate cell adhesion and proliferation. Further, we introduce electrical conductivity properties to the engineered fibrous GelMA scaffold through the incorporation of a choline-based bio-ionic liquid (Bio-IL) within the fabricated GelMA electrospun sheet, and photocrosslink the constructs in the presence of UV light.
Here, we performed mechanical characterization on these cardiopatches, which showed that the electrospun GelMA/Bio-IL scaffolds had a tunable elastic modulus based on GelMA and Bio-IL concentrations. The elastic modulus was shown to increase concomitantly with an increase in the concentration of Bio-IL incorporated onto fibrous GelMA mats. The Bio-IL incorporated GelMA fibers exhibited tunable electrical conductivity that increased consistently by increasing the concentration of Bio-IL. In addition, ex vivo experiments using excised rat abdominal tissues placed on electroconductive cardiopatches showed that the excitation threshold of these tissues decreased with and increasing concentration of Bio-IL. This test demonstrated the ability for fibrous scaffolds to propagate an electrical impulse and excite peripheral tissues. These electroconductive cardiopatches could be readily attached to the epicardial surface of the heart via photocrosslinking, which eliminates the need for sutures that could lead to further tissue trauma. In vitro cell studies performed by seeding cardiomyocytes and cardiac fibroblasts on the surface of our GelMA/Bio-IL cardiopatches demonstrated the scaffolds ability to support cell adhesion, and proliferation. Further, cells seeded on conductive cardiopatches exhibited a markedly improved contractile profile when compared with non-conductive patches due to the overexpression of the gap junction protein connexin 43 (Cxn43). Lastly, we were able to demonstrate the cardioprotective efficacy of these cardiopatches using a rat MI model where these electroconductive scaffolds were placed on the surface of the heart following permanent ligation of the left anterior descending coronary artery. Our histological assessment was indicative of comparatively lower thinning of the ventricular wall 3 weeks after induction of the experimental MI.
The results presented here demonstrate a new method to fabricate a highly conductive fibrous GelMA/Bio-IL cardiopatch with tunable mechanical and conductivity properties through incorporation of Bio-IL into a fibrous scaffold. Cardiopatches were also highly adhesive to wet tissue and was shown to be biocompatible in vitro using cardiomyocytes and cardiac fibroblast cells. Conductive cardiopatches were able to support the overexpression of Cxn43, necessary for cell-cell interaction. This conductive GelMA/Bio-IL cardiopatch has the potential to be implanted onto damaged site in the heart to provide mechanical stabilization to the injured myocardium and to restore electromechanical coupling at the site of MI.

An intrinsic ion sensitivity exceeding the Nernst-Boltzmann limit and an sp²-hybridized carbon structure make graphene a promising channel material for realizing ion-sensitive field-effect transistors with a stable solid-liquid interface under biased conditions in buffered salt solutions. We will present on the performance of graphene field-effect transistors coated with ion-selective membranes as a tool to selectively detect changes in concentrations of Ca²⁺, K⁺, and Na⁺ in individual salt solutions as well as in buffered Locke’s solution. Both, the shift in the Dirac point and transconductance could be measured as a function of ion concentration with repeatability exceeding 99.5%, and reproducibility exceeding 98% over 60 days. However, an enhancement in selectivity, by about an order magnitude or more, was observed using transconductance as the indicator when compared to Dirac voltage, which is the only factor reported to date. Fabricating a hexagonal boron nitride multilayer between graphene and oxide substrate further increased the ion sensitivity and selectivity of transconductance. These findings incite investigating ion sensitivity of transconductance in alternative architectures as well as urge the exploration of graphene transistor arrays for biomedical applications.

Nervous systems are remarkable for supporting stable animal behavior despite dramatic changes to neurons’ number and connectivity, yet many questions remain. For example, how do new neurons differentiate from stem cells and integrate into to existing circuits? Small invertebrate model organisms offer several advantages for studying this type of neural plasticity. Unlike organoids or cultured cells, the properties of the neural circuit in an animal can be directly related to behavior; however, most invertebrates show modest regeneration or lack well-developed transgenic techniques. Here we show that Hydra vulgaris, a millimeter-sized transparent cnidarian, with 12 neural subtypes shows simple sensory-motor transformations that are maintained despite dramatic changes to the numbers of neurons. The small size and deformable body make allow us to study neural activity in microfluidic platforms that permit chemical, electrical, and optical interrogation. By studying this type of self-renewal in a simple and tractable model organism we can discover fundamental principles that enable neural circuits to self-assemble and reorganize to maintain stable sensory-motor responses.

There has been significant advances in the synthesis and characterization of graphene and related two-dimensional atomic layers. For many applications, including biological, these materials need to be modified to introduce specificity, functionality and flexibility. The talk will focus on the generation of two-materials and approaches for their chemical and electronic modification. Various low dimensional structures that can result from 2D layers, such as quantum dots, ribbons and heterostructures, will be described showing how chemical and dimensional modifications can change their intrinsic properties. Interface engineering of these structures within matrices or as multilayers will be considered as opportunities to design and create new material interface systems. The talk will also address the usefulness of 2D materials in applications such as sensors, coatings and biological interfaces.

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First-generation nucleic acid analogs such as 2¢-deoxy-2′-fluoro-RNA (2′-F RNA), 2′-O-methyl RNA (2′-OMe RNA), and the phosphorothioates (PS-DNA/RNA) have remained staples of oligonucleotide modification strategies in the discovery and optimization of potential RNAi therapeutics.^1,2 The first FDA-approved siRNA drug, patisiran (Onpattro) contains eleven 2′-OMe ribonucleotides in the sense and antisense strands formulated in a lipid nanoparticle (LNP) delivery system.³ Despite their overall appeal in terms of enhanced nuclease resistance, increased pairing stability (2′-F/OMe RNAs) and safety, newer analogs are needed to further improve the therapeutic potential of unformulated siRNAs like GalNAc conjugates. The search for oligonucleotides with therapeutic efficacy has given rise to an expanding universe of analogs, including the second-generation 2'-O-(2-methoxyethyl)-RNA (MOE-RNA)⁴ and the third-generation bicyclic nucleic acids (BNAs or LNAs).⁵ The key enzyme in RNA interference (RNAi) is Argonaute 2 (Ago2), a dynamic multi-domain enzyme that binds multiple regions of the guide (antisense) and passenger (sense) siRNAs. Considering the complexity of protein-RNA interactions in RNA interference, it is imperative to expand the process of discovery and optimization of modified siRNAs to new analogs.⁶ We have explored the potential benefits for efficacy of incorporating into siRNAs sugar modifications like N-methylacetamide (NMA), 5′-E-vinylphosphonate (5′-E-VP) and, more recently, xeno-nucleic acid (XNA)⁷ residues such as glycol nucleic acid (GNA),⁸ altritol nucleic acid (ANA),⁹ and threofuranosyl nucleic acid (TNA).¹⁰ The talk will summarize siRNA modification strategies with an emphasis on regiospecific interactions between oligonucleotide and Ago2 and how structural insights based on crystallographic data for modified RNAs alone and in complex with Ago2 from molecular modeling studies can guide the choice of chemical modification at a given position of siRNA for optimizing its therapeutic efficacy.
1. Crooke, S. T.; Witztum, J. L.; Bennett, C. F.; Baker, B. F. Cell Metab. 2018, 27, 714-739.
2. Shen, X.; Corey, D. R. Nucleic Acids Res. 2018, 46, 1584-1600.
3. Sheridan, C. Nat. Biotechnol. 2017, 35, 995-997.
4. Teplova, M.; Minasov, G.; Tereshko, V.; Inamati, G.; Cook, P. D.; Manoharan, M.; Egli, M. Nat. Struct. Biol. 1999, 6, 535-539.
5. Pallan, P. S.; Allerson, C. R.; Berdeja, A.; Seth, P. P.; Swayze, E. E.; Prakash T. P.; Egli, M. Chem. Comm. 2012, 48, 8195-8197.
6. Egli, M.; Manoharan, M. Acc. Chem. Res. 2019, 52, 1036-1047.
7. Anosova, I.; Kowal, E. A.; Dunn, M. R.; Chaput, J. C.; van Horn, W. D.; Egli, M. Nucleic Acids Res. 2016, 44, 1007-1021.
8. Schlegel, M. K.; Foster, D. J.; Kel’in, A. V.; Zlatev, I.; Bisbe, A.; Jayaraman, M.; Lackey, J. G.; Rajeev, K. G.; Charisse, K.; Harp, J.; Pallan, P. S.; Maier, M. A.; Egli, M.; Manoharan, M. J. Am. Chem. Soc. 2017, 139, 8537-8546.
9. Ovaere, M.; Sponer, J.; Sponer, J. E.; Herdewijn, P.; Van Meervelt, L. Nucleic Acids Res. 2012, 40, 7573-7583.
10. Schöning, K.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.; Eschenmoser, A. Science 2000, 290, 1347-1351.

Cancer is a complex genetic moving target that requires a therapeutic modality capable of selectively targeting all driver oncogenes combined with the inherent ability to undergo pharmaco-evolution to target new mutations at pace with the cancer cell mutation rate. siRNA RNA-interference (RNAi) therapeutics fulfill these criteria and have great potential to treat cancer and other human diseases. However, due to their 14 kDa size and 40 negative anionic charges, siRNAs have no bioavailability to overcome a billion years of evolutionary defenses to prevent RNAs from crossing the endosomal lipid bilayer to enter the cytoplasm. Consequently, developing anti-cancer RNAi therapeutics requires targeted delivery to malignant cells and paradigm-shifting technology to enhance endosomal escape into the cytoplasm, the critical rate-limiting delivery step. Our research is focused on solving these problems by synthesis of bioreversible charge neutral RNAi prodrugs, called short interfering RiboNucleic Neutrals (siRNNs), and synthesis of novel hydrophilic-masked, hydrophobic endosomal escape domains (EEDs). For targeting domains, we have focused on conjugating antibodies to siRNN RNAi triggers to generate Antibody-RNAi Conjugates (ARCs).

Reduction sensitive linkers have the potential to transform the field of drug delivery due to their ease of use and selective cleavage in intracellular environments. However, despite their compelling attributes, using reduction sensitive linkers for biomolecule conjugation reactions is still challenging because linkers have not been developed that can efficiently modify aliphatic amines and release them rapidly and completely after reduction. Developing reduction sensitive self-immolative linkers for aliphatic amines has been challenging due to their poor leaving group ability and high pKa values. Here we present a traceless linker (TRAILER), which can for the first time modify aliphatic amines and release them rapidly and quantitatively after disulfide reduction. We show here that TRAILER can reversibly modify the lysine residues on the Cas9 protein, with the cell penetrating peptide Arg₁₀, and was able to generate a self delivering Cas9 RNP that could edit cells without transfection reagents. TRAILER has numerous applications in biotechnology given the ubiquitous presence of aliphatic amines on small molecule and protein therapeutics.

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Non-coding ribonucleic acids (ncRNAs) are a diverse group of RNA molecules that are not translated into proteins following transcription. Mainly, microRNAs and long non-coding RNAs (lncRNAs) are predominant in this group. Aberrant expression of these ncRNAs has been observed in various pathological conditions including cancer. Especially, aberrant expression of microRNAs and the associated pathology has been extensively studied in cancer. Hence, targeting microRNAs has been considered a new generation of molecularly targeted therapy for various diseases. While downregulation of cellular microRNAs can be supplemented by delivering microRNA mimics, functional knock down of microRNAs that are overexpressed in diseases is a challenging task. The secondary structure of microRNAs determines how to functionally inhibit the translation of target mRNAs. MicroRNAs with even a few base homologies to the target can functionally regulate mRNA degradation/translation. The secondary structure of microRNA is crucial for recognizing target mRNA for functional effect. Since the double stranded RNA is an important intermediate in the biogenesis process of matured microRNA, delivering antisense microRNA for functional knock down of endogenous microRNAs needs a careful design. Especially challenging is their site-targeted delivery. We study antisense microRNAs with various structural determinants (mere homology, end loops, base pair modifications, artificial linkage, etc.,) by molecular dynamics of the duplex formation at 1 micro second and beyond, for the evaluation of the therapeutic effect of microRNAs in various cancer models (GBM: antisense miR-21 and antisense miR-100; Breast cancer antisense miR-21 and antisense miR-10b; Hepatocellular carcinoma: antisense miR-21, antisense miR-10b and antisense miR-100). In this presentation, we summarize how structural modification in antisense microRNAs can be used for potential applications in cancer therapy, either microRNA targeting as a monotherapy, or for improving clinical chemotherapy.

We have recently developed a CRISPR powered, electronic biosensor termed, CRISPR Chip, capable of detecting the binding interactions between nuclease deactivated CRISPR with its target DNA sequence. CRISPR-Chip is a graphene field effect transistor (gFET) where the
surface of the graphene is functionalized with nuclease deactivated CRISPR RNA-guided ribonucleoproteins (dRNPs). dRNPs within the CRISPR-Chip construct interact with their target sequence by scanning the genomic sample until they find and bind to their target. The selective hybridization of the target DNA to the dRNP complex modulates the graphene’s conductivity and results in a detectable change in electrical signal output which can be measured with a simple handheld reader. CRISPR-Chip was able to detect dRNP binding to two commonly deleted exon target sequences within the human dystrophin gene, without amplification. CRISPR-chip is the first example of CRISPR-powered electronic transistors that harnesses the search function of CRISPR/Cas9 and the ultra-sensitivity of graphene-based nanoelectronics to enable a complete label-free and amplification-free DNA biosensor.

MicroRNAs are emerging as a novel diagnostic and therapeutic targets of human pathologies. Since the expression levels of various microRNAs are dysregulated in different cellular diseases, including cancer, understanding structural dependency of microRNAs for their biological functions is essential for designing synthetic oligos with various base and linkage modifications to develop high-sensitive diagnostic devices, and to use them in therapeutic applications. Here we use a label-free Raman spectroscopy to understand the structural difference of a selective microRNA, the microRNA-21, by hybridizing it with complementary RNA, DNA, and DNA with multiple base mutations for analysis. To verify the spectral changes induced by hybridization, the spectral profiles obtained from single-stranded microRNA-21 mimic, and RNA and DNA oligos complementary to microRNA-21 were compared with microRNA-21 in the form of hybrid and mixed with complementary oligos in solutions. The spectral changes were also analyzed for the concentration of complementary DNA with single, double and triple base mutations to understand Raman specific detection signal that can specifically distinguish different structural variants within a mixture. The analysis of the results identified prominent spectral features around 930 cm^-1, 1017 cm^-1 and 1068 cm^-1 for C-C and C=O stretching of the ribose ring. Similarly, the broad spectral features including a sharp band and a shoulder in the area of 1350-1380 cm^-1 were attributed to be adenine (C8–H and C2–H out of plane bending) and guanine (C8–H bending and C8–N stretching) bases. A band at 1418 cm^-1 was assigned to be N=C–H bending modes from adenine, guanine and cytosine bases of RNA sequence. While analyzing the intramolecular structure in RNA, we also analyzed the DNA spectrum of Raman bands at 841 cm^-1, 874 cm^-1 and 930 cm^-1 which were identified to be C2’-endo (B-DNA) conformation of DNA. In addition, we identified that the Raman band around 785 cm^-1 to be the phosphodiester bond of the backbone linking 5’ phosphate of nucleic acid with 3’ OH of the other nucleotide. This study successfully utilized label-free Raman spectroscopy to differentiate structural features such as intermolecular (ssRNA and ssDNA) and intramolecular (RNA-RNA and RNA-DNA hybrid) interaction which can be utilized for developing biosensors to quantify microRNAs in clinical samples and to design therapeutic microRNAs with strong functionality for the treatment of cellular diseases.

Upon systemic administration of nanoparticles used in drug and gene delivery, they acquire a “biological identity” upon adsorption of plasma proteins. The protein corona layer has significant implication in the biodistribution, target specific localization, and toxicity of nanoparticle-based delivery systems. Additionally, specific enrichment of proteins may allow for an endogenous and more efficient approach for targeted delivery.
Using a wide ranging of lipid nanoparticle (LNP) formulation where individual lipid composition was varied, we have shown specific protein enrichment pattern and differential biodistribution and tumor-specific delivery of small interfering RNA (siRNA). For instance, DOTAP functionalized LNPs showed vitronectin enrichment and enhanced in vivo tumor delivery of the nanoparticles to αvβ3 expressed tumors and endothelial cells as compared to αvβ3 negative tumors.

Current advances in RNA-based technologies such as therapeutic gene editing, mRNA therapeutics, and vaccines have produced strong interest and attention in safe and effective nonviral vectors for nucleic acid delivery. Nevertheless, the comprehensive application of nucleic acid therapeutics is still limited by the need for better delivery systems. Thus, the analysis of these delivery materials can be advanced to offer sharper insights into SAR learnings and provide a clearer picture of their therapeutic potential. Lipid- and polymer-based materials can be regarded as the most prominent partners for nucleic acid delivery, providing in vivo transfection. Over the past few decades, a variety of cationic lipids and polymers have been synthesized, evaluated and applied as successful delivery vehicles for nucleic acids. Here, I will discuss some of the challenges for clinical translation of mRNA-based therapeutics and give examples of the biomaterials and delivery strategies we are investigating for the application of mRNA delivery.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases, leading to cirrhosis, liver cancer and cardiovascular diseases, and is the important stage to cure the diseases without liver transplantation [1]. Until now, any effective drugs/treatments have been established due to the improper in vitro cell-based assays and animal models, misleading the results to study the disease mechanisms. A gut-liver axis (GLA) might play a critical role in NAFLD by disrupting intestinal barriers towards liver damages and fibrosis, and could be a good candidate model to study NAFLD in vitro. However, the conventional cell-culture settings did not allow recapitulating inter-tissues interactions like GLA. On the other hand, the recent Organs-on-a-Chip technology may offer recapitulating such interactions by utilizing microengineering/fluidic technology [2]. Here we report a gut-liver on a chip (iGL Chip) enabling the precise closed-loop medium circulation to represent GLA as an in vitro NAFLD model. We demonstrated to culture gut and liver cell lines (CaCO2 and HepG2 cells, respectively) under the medium recirculation, perturb with free fatty acids (FFAs) for cells to initiate NAFLD process, and monitor cellular responses in a chip.

The microfluidic device in iGL Chip composed with three polydimethylsiloxane (PDMS) layers, i.e., from top to bottom, perfusion, thin membrane, and control layers. To incorporate precise pressure-driven micro valves and pneumatic pumps utilizing thin elastic membrane, the part of a perfusion channel of the pneumatic pump has semicircular shape for the complete seal by deformed membrane. The designed device was fabricated by the combination of the simulation based-grayscale lithography and the softlithography process [3].

It is well-known that PDMS has the critical issue of an absorption of small hydrophobic molecules, and might cause FFA absorption. To reduce the absorption and improve cell growth on PDMS, PDMS surface was treated with n-dodecyl-D-maltoside (DDM) amphiphilic molecule, followed by Matrigel extracellular matrices mixture. The DDM coating reduced the background of adipoRed lipid fluorescent staining about 20% compared to bare PDMS. Next HepG2 cells and CaCO2 cells were separately introduced into their corresponding cell-culture chambers with 1.0×10⁶ cells/ml. The circulation flow (15 nL/min) was actuated by the micropump after 12 hours of cell attachment. For 5-7 days of cell culture, both cells reached to the full confluence in the cell culture chamber area. The effect of coating method on cell attachment and viability were tested by the Calcein-AM living-cell staining. The cell viability of DDM/Matrigel coating was similar with those of only Matrigel coating. The DDM/Matrigel coating could improve cell culture condition with the reduction of molecules absorption.

Next, co-culture of HepG2 and CaCO2 cells in a iGL Chip was demonstrated to investigate the effects of medium recirculation flow. Although HepG2 cells showed comparable cell viability among tested conditions, CaCO2 cells under flow conditions had higher cell viability than that of static conditions. Finally, to observe the cellular response after FFAs treatments, HepG2 and CaCO2 cells were treated with FFAs-contained medium (oleic acid : palmitic acid = 2:1) and kept under recirculation flow for 24 hours to investigate whether NAFLD-like cellular phenotypes. The intercellular lipid in HepG2 cells was increased with the increase of FFA concentrations, resulting the reduction of cell viability, on the other hands, CaCO2 cells did not change their viability after FFAs treatments. These results suggest the iGL Chip represented the initial state of NAFLD and has potential for disease modelling towards drug developments for NAFLD.

[1] B. Li et al., Can. J. Gastroenterol. Hepatol, 8 (2018), 2784537
[2] S. Ahadian et al., Adv. Healthcare Mater. 7 (2018), 1700506
[3] K. Kamei et al., RSC Adv., 7 (2017), pp.36777−36786

Defective models, such as gene-knockout cell model, gene-knockout mouse model, or immune-system-inhibited nude mouse model, have played critical roles in biological studies. We extrapolated this kind of idea into the field of organ-on-a-chip research, and proposed a defective organ-on-a-chip strategy, which we believe would find wide applications in the field of pharmaceutical and biomedical researches. To demonstrate this strategy, we developed an immuno-liver-chip featuring T cells, and realized preclinical drug-hepatotoxicity-typing by exploiting defective liver chips. In detail, we removed T cells, Kupffer cells, Stellate cells, and the artificial bile duct from the immuno-liver-chip, respectively, thus deriving four distinct defective liver chips. We then measured the hepatotoxicity of a same drug with these immuno-liver-chip series. By analysis of the difference between the measured hepatotoxicity, we can identify the role of different types of hepatic cells in the drug hepatotoxic process and therefore deduced the mechanism of the drug hepatotoxicity. As a demonstration, we used defective liver chip strategy to successfully discriminate the hepatotoxicity mechanism of four classical drugs, Abacavir, Ethynyl estradiol, Paracetamol, and Sulfamethoxazole.

Chronic kidney disease is a public health problem affecting 850 million people worldwide, 37 million people in the US adult population, and is the 9^th leading cause of death according to the Centers for Disease Control and Prevention. Until recently there has been a lack of human in vitro models that recapitulate critical aspects of kidney physiology, mimic the unique complexities of specific nephron segments, model disease heterogeneity or assess reparative mechanisms in response to injury. The lack of in vitro models, coupled with numerous challenges in available animal models for modeling most human kidney diseases, has hindered the development of safe and effective therapies. This is in part because microfluidic flow is so essential to kidney structure and cellular function. In response to this critical unmet need, we and others have developed three-dimensional flow directed “kidney-on-a-chip” microphysiologicsl systems (MPS) populated with a variety of human kidney cells, with functional characterization of key component structures of the proximal tubule, the peritubular microvascular network, and more recently the glomerular filtration barrier. Use of our kidney-on-a-chip MPS platform has been accompanied by the development of multiple reproducible data readouts, including immunocytochemistry, advanced microscopy, deployment of protein and microRNA biomarkers and mRNA gene expression data. The kidney MPS is characterized by precise control of cellular composition, extracellular matrix, vascular and tubular geometry and flow. Our integrated approach for in vitro disease modeling includes differentiating human kidney cells and organoids from diseased patient-derived inducible pluripotent stem cells (iPSC), CRISPR gene editing, single cell transcriptional profiling and engineering living human kidney vascular networks. We have demonstrated success in delineating novel molecular drivers of several disease processes and drug induced nephrotoxicity. Our in vitro on chip models recapitulate critical aspects of kidney physiology, assess the mechanisms and response to injury, have established translatable biomarkers and target engagement readouts, and test reparative mechanisms, all of which can substantially enhance therapeutic discovery and evaluation for a varity of kidney diseases.

The human ‘Organ-on-a-Chip’ (Organ Chip) microfluidic culture technology was originally developed as a tool to provide better predictive models for drug efficacy and toxicity. We have developed 2-channel Organ Chip devices that recapitulate tissue-tissue interfaces, vascular perfusion and organ-level functionality seen in vivo by incorporating multiple cell types, mechanical cues (e.g., breathing or peristalsis-like motions), and fluid flow, as well as immune cells. Using this approach, we have created Organ Chip models of over 10 different human organs, as well as rat and dog Liver Chips. We have also developed a robotic Interrogator instrument that can link up to 10 different Organ Chips to form a ‘Human Body-On-Chips’ platform, which we have used in combinational with computational modeling to carry out predictived in vitro-to-in vivo extrapolation (IVIVE) of human drug pharmacokinetic and pharmacodynamics (PK/PD) parameters in vitro. Using these approaches to carry out human experimentation in vivo, we have not only addressed key challenges relating to drug PK/PD, toxicity, and efficacy, we also have gained new insights into human physiology and disease mechanisms, and have been able to model various diseases and rare gene disorders using patient-derived cells. Thus, Organ Chips offer a powerful new approach to confront challenges relating to mechanistic pathophysiology as well as drug development and personalized medicine.

Cancer is currently the second leading cause of death in the United States, claiming the lives of over half of a million Americans annually. Poor patient prognosis and treatment outcomes are in large part due to the biological complexities of this devastating disease. Improved treatment and early diagnosis have yielded a steady decrease of cancer-related mortalities, but metastatic cancers are still deadly due to a lack of effective targeted treatment options. Upon tumor formation at the primary sites, metastasis progresses through a stepwise cascade of events, in which the key initial steps include stromal desmoplasia/fibrosis, immune response, tumor invasion and cancer cell escape into the vasculature (i.e. intravasation). Tumor cells adopt an aggressive phenotype through continuous interactions with their surrounding microenvironment (TME) and the multitude of physiochemical signaling cues that emerge from their surrounding extracellular matrix (ECM) and resident stromal cells. To that end, the complexities of the native TME and the limitations of conventional in vitroplatforms and in vivotumor models pose a non-trivial challenge in identifying the key molecular and cellular signaling cues involved in the dynamic tumor-stroma crosstalk as potential therapeutic targets. In the past few years, microengineering technologies have been widely utilized in various scientific disciplines, from physics and chemistry to biology. These technologies have proved immensely beneficial for the fields of experimental biology and medicine through development of miniaturized biomimetic living tissue systems for cellular- and molecular-based studies. In addition, these technologies have gained significant attention in their ability to precisely control cell-microenvironment interactions to better understand the mechanisms of human disease progression, particularly cancer. In this seminar, I will present the multidisciplinary research focus of our laboratory at ASU on the integration of microscale technologies, advanced biomaterials and biology to develop the next generation ex vivo on-chip tumor microenvironment models to study cancer progression at the earliest stages of the metastatic cascade. I will specifically discuss our recent efforts and findings on engineering of organotypic breast and brain tumor microenvironment models to dissect the role of stromal cells (e.g. cancer associated fibroblasts) on tumor cell invasion, intravasation and vascular homing.

Impediments to drug discovery include increasing costs (over 10 billion USD per drug), long period of development (more than 10 years), insufficient drug efficacy, and serious side effects. These issues can largely be attributed to the current improper approach to pre-clinical testing that involves the use of experimental animals, often yielding misleading results because of variation in responses due to species difference. In addition, the use of experimental animals has become restricted in many countries due to ethical issues. Therefore, drug development by pharmaceutical companies is a high-risk endeavor and new approaches for drug discovery are urgently required.
The microphysiological system (MPS, which is also termed organs/body-on-a-chip) has considerable potential for use in drug development and toxicological testing. MPSs aim to recapitulate in vivo physiological and pathological conditions in a chip without the use of actual organs, similar to an in vitro human model. Compared with conventional animal models, MPSs are advantageous for predicting drug efficacy and toxicity in humans, obtaining more quantitative results, shortening experimentation time, improving the reproducibility and cost-effectiveness, and reducing the use of animals. The MPS platform could become an alternative to animal testing.
However, the current MPS platforms have several issues that need to be resolved. Most MPS platforms still possess a one-way flow stream or a few branches on a chip, so they cannot completely represent the inter-tissue interaction that occurs through the blood circulation system. Some studies have utilized external rotary peristaltic pumps for cell-culture medium circulation, but they require a large amount of medium, and drugs, metabolites, and products secreted from cells could be diluted and lost as a result of this process. Notably, most of the previously developed MPS platforms can only examine a set of samples on the chip, and do not have the capability to compare samples next to each other within a device. Furthermore, it is still challenging to directly integrate micropumps in a device, because the existing fabrication methods are not simple and reliable. Therefore, it is important to develop new microfabrication technologies capable of integrating pumps into a device to minimize medium volume and sample loss.
Moreover, it is also important to integrate an in situ sensor to monitor cell or tissue behavior after administering the treatments on a chip. Majority of the current cell monitoring methods used in the MPS platform are based on microscopic imaging with cell staining. However, this approach often requires cell-labeling materials that could perturb cells, influencing the drug treatment or killing the cells. Additionally, this approach only permits end-point experiments and there is loss of information over time. Accordingly, it is necessary to integrate in situ and non-invasive cell monitoring systems to obtain “living cell” information.
In the present study, we introduce an interdisciplinary approach comprising aspects of stem cell biology, material science, and micro/nano-engineering that is designed to solve the aforementioned issues. We present some applications, such as in vitro recapitulation of anti-cancer drugs on the heart as well as in vitro modeling of non-alcoholic fatty liver diseases. We envision that this technological development will allow a precise recapitulation of human physiology and pathology, which will enhance our understanding of the mechanisms underlying various diseases for which effective treatments remain elusive, and will help identify new therapeutic drugs.

Brain metastasis (BM) is a leading cause of mortality in patients with non-small cell lung cancer (NSCLC). However, the molecular mechanisms underlying BM of NSCLC remain largely unknown because of the lack of models to accurately investigate such a dynamic and complex process. Here we developed a multi-organ microfluidic chip as a new methodological platform to study BM. The chip consisted of two bionic organ units – an upstream “lung” and a downstream “brain” characterized by a functional “blood-brain barrier (BBB)” structure, allowing real-time visual monitoring of the entire BM process, from the growth of primary tumor to its breaking through the BBB, and finally reaching the brain parenchyma. The chip was then applied for the BM research where we first demonstrated that the protein expression of Aldo-keto reductase family 1 B10 (AKR1B10), which was identified by proteomics profiling, was significantly elevated in lung cancer BM. Silencing AKR1B10 in brain metastatic tumor cells suppressed their extravasation through the BBB in the in vitro Transwell model, in our ex vivo microfluidic chip, as well as the in vivo model of brain metastasis in nude mice. With the combined application of our new model and traditional research platform, it was suggested that our multi-organ microfluidic chip is a practical alternative to study BM pathogenesis, and AKR1B10 is a diagnostic biomarker and a prospective therapeutic target for NSCLC BM.

Hepatic tissue is a complex organ with very specific fluid flow characteritics. The adverse effects of chemicals and drugs are studied in vitro by conventional two dimensional monolayer cultures. 2D cultures results in loss of cell viability and decreased liver-specific functionality. In vitro models such as spheroid culture, sandwich culture and 3D printed tissues have been proposed to overcome these limitations. When culture systems are upgraded from 2D to three dimension (3D), the microenvironment also needs to be upgraded. We propose a 3D Printed hepatic lobule-Like bioreactor for in vitro Hepatotoxicity testing. The multi chamber in the perfusion device can hold functional hepatocytes and is nourished by flow conditions. Various flow conditions were simulated in silico to understand the fluid flow inside the device. Bifabricated tissues, either cell encapuslated or 3D bioprinted, placed inside the device receives fluid flow similar to liver lobule and are functional. The device can perform like a miniaturized disposable bioreactor for hepatotoxicity testing

In the scope of biomedical research, many advances have been made in the field of pulmonology with the use of multiple animal models and traditional cell culture experimentation. While these methods are useful when studying single cellular pathways or overarching biological processes, they often falter when studying the meticulous nature of human disease. In this work, we propose the development of a novel Lung-on-a-Chip device that would more accurately mimic the physiological boundaries of human alveoli than animal models or traditional in vitro studies. Utilizing multiple microfabrication techniques, we are developing a microfluidic device with an ultra-thin (25 µm) biodegradable porous silicon membrane. Current data suggests that human pulmonary epithelial and endothelial cells are viable and prolific on the proposed microfabricated silicon membrane in extended studies (14 days). Due to the biodegradability of the fabricated novel silicon membranes, it has been observed in long term studies that cells can remodel and degrade the porous silicon membrane. This degradation allows for physiological cellular contact between membranes mimicking a true blood gas exchange interface as observed in vivo. To further validate this model’s ability to recapitulate human physiology, we have begun to co-culture with immunological cell types and are monitoring for response to bacterial and tumor antigens. Preliminary data suggests that immunological cells are able to remodel the porous silicon membrane substrate and migrate across cellular boundaries in response to environmental cues as seen in vivo. Broadly, we believe that this model may be used to further characterize and study human pulmonary disease and drug toxicity.

Organ-on-a-chip (OOC) technology progresses rapidly and had attracted tremendous interest in research due to its capabilities of emulating in-vivo microenvironment and tissue structure. It has great potentials for pharmaceutical research development and precision medicine. Microfluidic technology and precision 3D cell printing are the two key enablers to make OOC. However, current OOC has some drawbacks including low throughput, inconsistency, high-cost, unmanufacturable. These issues has prevent wide use of OOC technology in industry scale.
In this work, we demonstrate an approach to standardize Organ-on-a-chip (OOC) fabrication and testing process. In this process, A inkjet printing system with special designed MEMS printhead has be built and employed to printing multiple types of cells and structural bio materials in a precise and repeatable way. In addition, a special designed trans well structure and removable wells are used to hold OCC with multiple replicas. This standardized OOC process is able to provide a standardized and reliable method for 3D multi-type cell co-culture with a integrated perfusion system which can adjust flow rate to provide stable shear-force microenvironment for better simulating human-like in vivo system including hepatic sinusoids, blood-brain-barriers (BBB), intestinal villi.

Analysis of C.elegans by droplet microfluidics has been widely used in study of locomotive behavior responses to neurotoxicity due to the capacity of high-throughput manipulating single cells. However, it has been difficult to manipulate droplets flexibly and actively on account of the limitation the dimension of individual C. elegans droplets. In this study, a novel MiDMS (Micro-injection Droplet Microfluidic System) was proposed, which consist of three parts: single C. elegans droplet generator, droplets drug micro-injection channels and drug-incubation observation array. Individual C.elegans droplets were produced initiatively by regulating the flow rates between oil and water phase as well as the concentration of C.elegans in suspension. Then, the drug solution was precisely injecting into each C.elegans droplet, which by electricity induced surface tension of droplet changing. In addition, the effect of neurotoxic Cu²⁺ on locomotive behavior of C. elegans was evaluated at single cell resolution. The results showed that the neurotoxicity induced behavioral disorder of the C. elegans was more obvious with the increase of Cu²⁺ concentration treatment time, and these dose-effect and time-effect relationship in MiDMS were similar as in petri dish. It will be providing a powerful platform for the study of the response of C. elegans to quantitative drug at single cell resolution.

A lab-on-a-chip diagnostic device for the point of care testing of non-communicable diseases has been developed considering the requirements of low cost, ease of use and diagnosis at every door-step. The device is capable of monitoring diabetes, chronic kidney and liver disorders, gout and oxidative stress-related diseases through simultaneous measurement of glucose, creatinine, bilirubin, uric acid and ascorbic acid from a single drop of body fluid. This device eliminates geographic, economic and rural barriers in healthcare and will revamp healthcare by providing access to affordable technology for better management of major non-communicable diseases.
The device consists of three parts, nanomaterial-based electrochemical sensors, a microfluidic chip and an electronic meter with read-out.
A disposable non-enzymatic, electrochemical creatinine sensor was developed using copper nanoparticles modified screen-printed carbon electrodes. The detection was based on the formation of soluble copper–creatinine complex at the applied potential. The sensor exhibited excellent response towards creatinine in the range of 6.25–378.5 μM at the physiological pH making it highly suitable for real sample analysis.
Non-enzymatic, electrochemical detection of bilirubin was carried out on disposable screen-printed carbon electrodes. The sensor exhibited excellent catalytic response towards bilirubin in the range of 5–600 μM at pH 8.5 making it highly suitable for real sample analysis. The sensor was successfully used to analyze albumin-bilirubin complex formation. The effect of ibuprofen on the unbinding of bilirubin-albumin complex was also demonstrated with the sensor.
Non-enzymatic, electrochemical detection of glucose was carried out on Pt-CuO-rGO modified SPE. The sensor exhibited excellent catalytic response towards glucose in the range of up to 27 mM in alkaline pH. A disposable non-enzymatic electrochemical sensor for AA was fabricated using copper hydroxide nanorods. The sensor works at very low potential with excellent linearity (0.0125–10 mM), sensitivity (268 µA/mM/cm²) and selectivity. The electrochemical detection of UA was carried out on activated SPCE in 0.1 M PBS. The sensor showed good linear response for UA in the range 100−1000 µM. A comparison of blood serum analysis results with those obtained from conventional clinical methods was highly correlative which further reiterates the potential of the developed sensors for POCT application.
Non-enzymatic electrochemical sensors were integrated into the microfluidic chip and tested with the indigenously developed programmable electronic meter. The device provides the preprogrammed potentials to the sensor electrodes which resulted in the oxidation of respective analyte on the working electrode. The voltage proportional to the reaction current was fed to the microcontroller from each analog front end. With the help of the calibration equation programmed into the microcontroller, the obtained voltage is converted into the corresponding analyte concentration and the results were displayed on the graphical LCD screen.
The developed LOC device is completely portable, handheld and can provide test results typically within a few minutes. The device is very simple to use and can be operated even by an individual with minimal technical training. Thus, the device will play a positive role in the prevention and management of non-communicable diseases namely diabetes, chronic kidney and liver disorders, gout and oxidative stress-related diseases, ensure better quality of life and also reduce the economic and social burden.

Recent outbreaks of the Ebola virus infection in several countries demand a rapid point-of-care (POC) detection strategy. This talk reports on an innovative pathway founded on electronic resonance frequency modulation to detect Ebola glycoprotein (GP), based on carrier injection-trapping-release-transfer mechanism and standard antibody-antigen interaction principle within a dielectric-gated reduced graphene oxide (rGO) field-effect transistor (GFET). The sensitivity of the current Ebola detection can be significantly enhanced by monitoring the device’s electronic resonance frequency, such as inflection frequency (f_i), where phase angle reaches maximum (θ_max). In addition to excellent selectivity, a sensitivity of ~36-160 % and ~17-40 % for 0.001-3.401 mg/L Ebola GP can be achieved at high and low inflection resonance frequencies, respectively, which are several orders of magnitude higher than the sensitivity from other electronic parameters (e.g., resistance-based sensitivity). Using equivalent circuit modelling on contributions of channel and contact, analytical equations for resonance shift have been generalized. When matching with the incoming ac measurement signal, electronic resonance from the phase angle spectrum is evolved from various relaxation processes (e.g., trap and release of injected charge at surface trap sites of channel-gate oxide and channel-source/drain interface) that are associated with a characteristic emission frequency. Using charge relaxation dynamics, a high-performance bio-FET sensing platform for healthcare and bio-electronic applications is realized through resonance shifting.

Oral cancer (OC) is currently one of the most wide-spread cancers and it is known to occur more often in men than women.[1] If not detected early, this cancer can be life threatening. Smoking, alcohol consumption, diet (red and processed meat, fried foods, chewing tobacco, human papillomavirus, and exposure to certain chemicals (e.g. sulfuric acid, asbestos, and formaldehyde) are some of the common reasons behind the onset of OC. Many techniques such as laser capture microdissection, visualization adjuncts, cytopathology, and biopsycan be for detection and monitoring of OC. These methods are time-consuming, expensive, labour intensive and invasive. Biosensors are, however, user-friendly, reliable and offer increased assay speed, high sensitivity and require low sample volumes.

Nanostructured metal oxides have recently aroused much interest as immobilizing matrices for development of biosensors since these materials provide desired orientation, better conformation and high biological activity resulting in enhanced sensing characteristics for oral cancer detection.[2–5] In this context, nanostructured oxides of metals such as zirconium, yttrium and hafnium have been found to show interesting functional, biocompatible, non-toxic and catalytic properties for oral cancer detection. These materials have been predicted to yield enhanced electron-transfer kinetics and strong adsorption capability and provide suitable microenvironments for the immobilization of oral cancer biomarkers e.g interleukin- 8 (IL-8), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF) and cytokeratin fragment-21-1(Cyfra-21-1) resulting in enhanced electron transfer and improved characteristics for OC detection. Among the various biomarkers, CYFRA-21-1 is a water-soluble proteinaceous biomarker and is a fragment of 40 kD of cytokeratin 19. [1] The cut-off concentration of CYFRA-21-1 in saliva for normal persons is 3.8 ng mL⁻¹ and patients have been found to have CYFRA-21-1 concentration as high as 17.46 ng mL^–1 in saliva. I will focus on the results of some the recent experiments obtained in our laboratories on nanostructured metal oxides based biosensors for non-invasive oral cancer detection.[3-5]

References

1.CYFRA 21-1 as a prognostic and predictive marker in advanced non-small cell lung cancer in a prospective trial, Martin J. Edelman1, Lydia Hodgson, Paula Y. Rosenblatt, Robert H. Christenson1, Everett Vokes, Xiaofei Wang, Robert Kratzke, and Cancer and Leukemia Group, J Thorac Oncol. 2012 April ; 7(4): 649–654
2.Nanostructured metal oxide-based biosensors,Pratima R. Solanki,Ajeet Kaushik,Ved V. Agrawal & Bansi D. Malhotra, NPG,Asia ,Materials ,2011, 3, 17–24
Biofunctionalized Nanostructured Zirconia for Biomedical Application: A Smart Approach for Oral Cancer Detection,2015 , Suveen Kumar, S. Kumar, Sachchidanand Tiwari, S. Srivastava, M. Srivastava, B.K.Yadav, S.Kumar, T.T.Tran, A.K.Dewan, A.Mulchandani, J.G.Sharma,S.Maji and Bansi Dhar Malhotra, Advanced Science ,2015, 2,1500048
4.Effect of Brownian motion on reduced agglomeration of nanostructured metal oxide towards development of efficient cancer biosensor,Biosensors and Bioelectronics,2018,102, 247-255
5.Biofunctionalized nanostructured yttria modified non-Invasive impedometric biosensor for efficient detection of oral cancer, Suveen Kumar ,Shweta Panwar,Saurabh Kumar ,Shine Augustine and Bansi D. Malhotra , Nanomaterials 2019, 9(9), 1190

Graphene has numerous applications in biomedicine as its unique physicochemical properties could be tuned to functionalize proteins, enzymes and other macromolecules for biosensor applications, or can be used as a therapeutic delivery vehicle for drug and nucleic acids delivery that can improve human life. Miniaturization of devices comes with a prerequisite need for precision control and selective placement of nanomaterials, which is still a challenge in most biosensing applications. In this study, we explore the potential of a graphene-based Field Effect Transistor (G-FET) device for glucose sensing. Specifically, we aim to selectively and precisely place monolayer of graphene on Si/SiO₂ substrates through the Langmuir-Blodgett technique (self-assembly process) and electrodes are fabricated on top of the graphene. Subsequently, the microchannels were integrated with the device and were functionalized with linker molecules and further immobilized with enzymes, which can catalyze glucose metabolism by direct electronic transport. Our study will highlight the potential use of graphene-based nanomaterials as a platform for developing devices that can be used for electrochemical biosensing, optical biosensing as well as the creation of graphene-metal nanoparticle hybrids for enhanced performance.

We report results of the studies relating to the synthesis of biphasic (monoclinic and tetragonal) nanodot zirconia (zero dimension with size < 4 nm) that has been utilized for the fabrication of electrochemical biosensing platform for the detection of CYFRA-21-1 biomarker; secreted in saliva samples of oral cancer patients. In the synthesis of nanodot zirconia, hydrothermal process was used at 180 ^oC for 24 h. The 3-aminopropyl triethoxy silane (APTES) functionalized nanodot zirconia (APTES/ndZrO₂) was electrophoretically deposited onto prehydrolyzed indium tin oxide (ITO) coated glass electrode at the potential of 14V for 120s. The monoclonal anti-CYFRA-21-1 (50 µg mL^-1) was drop-cast onto the obtained uniform thin film of APTES/ndZrO₂/ITO via EDC-NHS coupling. The non-specific binding sites were blocked using bovine serum albumin (BSA, 1 mg dL^-1). The X-ray diffraction (XRD), high resolution transmission electron microscopy (HR-TEM) and Fourier transforms infrared spectroscopy (FT-IR), were used for characterization of nanomaterials_. The electrochemical characterization and sensing response studies; cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques were used. The fabricated biosensing platform (BSA/anti-CYFRA-21-1/APTES/ndZrO₂/ITO) exhibited electrochemical response in the range of 2-20 ng mL^-1, sensitivity of 3.2 µA mL ng^-1cm^-2 and lower detection limit of 1.96 ng mL^-1. The observed electrochemical response of the BSA/anti-CYFRA-21-1/APTES/ndZrO₂/ITO was in good agreement with the results obtained through the enzyme linked immunosorbent assay technique (ELISA) in saliva samples of oral cancer patients.
References:
1. Kumar, S.; Kumar, S.; Tiwari, S.; Srivastava, S.; Srivastava, M.; Yadav, B.K.; Kumar, S.; Tran, T.T.; Dewan, A.K.; Mulchandani, A., Biofunctionalized nanostructured zirconia for biomedical application: A smart approach for oral cancer detection. Advanced Science 2015, 2.
2. Rajkumar, K.; Ramya, R.; Nandhini, G.; Rajashree, P.; Ramesh Kumar, A.; Nirmala Anandan, S., Salivary and serum level of cyfra-21-1 in oral precancer and oral squamous cell carcinoma. Oral diseases 2015, 21, 90-96.
3. Kumar, S.; Kumar, J.; Sharma, S.N., Nanostructured titania based electrochemical impedimetric biosensor for non-invasive cancer detection. Materials Research Express 2018, 5, 125405.

Inkjet printing technology encompasses the generation, control and deposition of 1-100 picolitre liquid drops. Besides graphic printing applications, new opportunities for inkjet printing are starting to be exploited commercially in the manufacture of high value, high precision products. The applications include the fabrication of 3D biological tissues and organs for regenerative medicine as well as flexible electronics such as biosensors, solar cells, and light emitting diodes. This talk present inkjet-based cell printing technology and its applications to regenerative medicine and tissue engineering. I will show how living cells are ejected from micron-sized nozzle with single-cell level accuracy and how multiple types of cells can be patterned into 2D and 3D biological structures with high-resolution. Then, our recent studies on the applications of 3D inkjet bioprinting, including high-throughput screening for personalized medicine, 3D alveolar barrier models, engineered skin substitutes will be presented. We believe that drop-on-demand, cell-accuracy inkjet printing is a versatile tool to fabricate complex tissues for drug discovery, disease model, and toxicity.

According to the World Health Organization, 9.6 million people worldwide died from cancer in 2018 (an estimated 1 in 6 global deaths) with a total economic impact of $1.16 trillion per year. Deaths due to prostate and breast cancer are often the result of metastasis to bone. Currently, there are no viable markers for metastasis. We report the development of novel in vitro models of breast cancer and prostate cancer metastasis through bone tissue engineering approaches. The tissue engineered scaffold uses amino acid modified nanoclay to develop polymeric nanocomposites. This scaffold uses biomimetic mineralization of hydroxyapatite inside nanoclays galleries, mimicking remodeling human, characterized by low Ca/P stoichiometry, a niche to which cancer cells migrate. Seeding the tissue engineered bone scaffolds with prostate and breast cancer cell lines, after bone formation, led to the creation of tumoroids of cancer. Bone metastasis is characterized by unique and complex biochemical, morphological, and genetic changes. The overall nanomechanical response of cancer tumors is representative of the complex changes at progression of metastasis. Here we report static and dynamic evaluation of nanomechanical properties of cancer cells during disease progression at the metastatic site. We report a significant softening of cancer cells as disease progression occurs at metastasis. We also examine the cytoskeletal changes that result in the observed mechanical behavior and its evolution at metastsis. The significant reduction in elastic modulus of cancer cells was attributed to reorganizetion of cytoskeletal elements. We report here the use of mechanics as a potential new marker for cancer metastasis progression.

Development of Molecular Imaging Smart Probes Based Technologies for High Resoluted Images of Prostate Cancer in MRI
Jebasingh Bhagavathsingh,^1* Divya Rajendran,¹ Sundar Manoharan,² Jannet Vennila,³ Nagabhushan Vellala,^4
1Department of Chemistry, Karunya Institute of Technology and Sciences, Coimbatore-641114, INDIA
²Department of Nanoscience and Technology, Satyabama Institute of Science and Technology, Chennai, INDIA
³Department of Biotechnology, Karunya Institute of Technology and Sciences, Coimbatore-641114, INDIA
⁴Department of Biochemistry, Microbiological Laboratories Research Service (P) Ltd., R. S. Puram, Coimbatore, INDIA
Email: [email protected]

Abstract:
Currently much attention has been devoted to the image quality emerging from any of the imaging modalities such as MRI, PET, SPECT, Optical Imaging, etc., There are two major approaches to achieve the high-resolution images from the existing imaging modalities such as i) by tuning the hardware parameters like magnetic field strength, signal-to-noise ratio, matrix size, field of view, etc., and/or ii) by using smart or responsive probe as an imaging agent for the in-vivo imaging of biochemical or physiological abnormalities as the origin of the diseases.¹ It is well-known that much of in-vivo experiments were unsuccessful after the successful standardization of in-vitro experiments due to the competitive in-vivo targets and/or administration of non-specific imaging agents.² Therefore, molecular imaging probe allows earlier with more personalized diagnosis and also facilitates to understand their molecular factors underlying pathological process using the smart behavior of imaging agents. Smart or responsive probe often provides well-resolution images from MRI detectable responses depending on the specific parameter such as pH, the concentration of ions or metabolites, temperature, enzyme sensitive, etc., at the molecular or cellular level.³ The multimeric paramagnetic chelates have proven with promising results in diagnosis, risk stratification, localization and staging of clinically significant prostate cancer. Herein we report the development of sugar capped biphenolic systems as enzyme sensitive smart probes and its Gd(III) chelates for high-resolution MRI applications. The sugar capped dopamine intermediate is synthesized by the glycosylation of protected dopamine using acetobromogalactose. The as-prepared intermediate is reacted with DOTA-Monoamide moiety for the paramagnetic metal ions chelation. Upon the removal of protecting groups, the high polar compound was purified by flash chromatography followed by the complexation with lanthanides. Upon the cleavage of the sugar moieties by the enzyme β-galactosidase, the relaxivity is significantly enhanced due to the formation of macromolecular adducts with the metal centers during the self-assembly process. The aggregation of Gd(III) based imaging agents provides the high-resolution MR Images at the conventional MRI (1.5 Tesla).
Reference
Wahsner, J.; Marthinos, A. A.; Gale, E. M.; Rodriguez, A. –R.; Caravan, P. Chemistry of MRI Contrast Agents: Current Challenges and New Frontiers. Chem. Rev. 2019, 119, 2957-1057.
Bonnet, C. S.; Toth, E. Smart Contrast Agents for Magnetic Resonance Imaging. Chimia 2016, 70, 102–108.
Hussain, T.; Nguyen, Q. T. Molecular Imaging for Cancer Diagnosis and Surgery. Adv. Drug Deliv. Rev. 2014, 66, 90-100.

Prostate cancer in its late stage is likely to metastasize to bone. However, a reliable marker for bone metastasis does not exist to date. The purpose of this study is to test the feasibility of using Raman spectroscopy for diagnosis of bone metastasis. MDA PCa 2b cells (metastatic prostate cancer cell from bone) were grown in the previously developed bone mimetic scaffolds. Our results indicate distinct time-dependent differences of metastatic prostate cancer cells grown in the scaffolds. Raman spectroscopic features in lipid, protein and DNA regions of these cells represent valuable diagnostic information which can be potentially translated into a clinic through fiber optic Raman probes. We also used a variety of novel spectral and statistical methods such as PCA to classify the event of bone metastasis. Overall, we demonstrate the feasibility for using Raman spectroscopy along with the powerful data analysis tools for next generation metastasis diagnosis and prediction.

Image guided delivery of therapeutics offer a novel approach to correlate therapeutic outcome with the level of delivered agents. Especially in neurological disorders, including GBM, the biological barriers of the brain present a major challenge in achieving adequate delivery of therapeutic drugs for treatment. Hence it is important to develop novel delivery agents with theranostic potentials to address these issues. We explore here the potential of the nose-to-brain direct transport pathway to bypass the blood-brain barrier (BBB) to enable targeted delivery of theranostics while implementing imaging as a complementary strategy to monitor the delivery. We adopted polyfunctional gold-iron oxide nanoparticles (polyGIONs) surface loaded with therapeutic miRNAs as a platform. This nanoformulation allows for monitoring the delivery by multimodality imaging. We used the microRNA delivery to presensitize GBM cells to the systemically delivered chemotherapy drug temozolomide (TMZ). We studied this approach in vivoby multimodality molecular and anatomic imaging to monitor, 1) nanoparticle delivery, 2) trafficking, and 3) treatment effects. For efficient loading of microRNAs, we used GIONs coated with b-cyclodextrin-chitosan (CD-CS) hybrid polymer. We used PEG-T7 peptide conjugated using CD-adamantane host-guest chemistry as a targeting agent. The resultant polyGIONs showed efficient miRNA loading, enhanced serum stability, and functional release in cells. For in vivointranasal delivery, we used orthotopic human xenograft mice model. Intranasal delivery resulted efficient accumulation of miRNAs in mice GMB. We used optical fluorescence, MRI, CT and Photoacoustic imaging to demonstratein vivodelivery and therapeutic efficiency in mice. We used optical bioluminescence imaging to measure therapeutic response of tumor to microRNA and TMZ combination therapy. Overall, predelivery of microRNAs sensitize GBM to TMZ and significantly increased survival rate of mice, compared to the other control groups. Once translated clinically, this novel theranostic nanoformulation and its associated intranasal delivery strategy will have a strong prospective to potentiate the effect of TMZ treatment in GBM patients.

MRI is currently used as a modality to detect, characterize, and monitor disease entities. Despite its inherent high soft tissue resolution and lack of ionizing radiation, the modality can be further enhanced to function as a biomarker for specific disease processes. This invited talk will highlight key advances that can further the use of MRI in the era of precision medicine and allow it to function as a useful biomarker, eventually to a single molecue resolution.

Breast cancer, in the late stages, disseminates to the bones, resulting in bone metastasis. Currently, there is no cure available for bone metastasis as the underlying mechanism of the processes that occur at the cellular level is poorly understood due to the failure of animal models and the scarcity of human bone metastasized samples as most patients with advanced stages of cancer are already in palliative care. Therefore, it is imperative to develop translational disease models to find a cure for cancer by elucidating disease mechanisms at the cellular and molecular level. In recent years, three-dimensional (3D) culture models have attracted substantial attention due to their ability to recapitulate in vivo characteristics, leading to altered gene expression, cytokine secretion, and improved drug response. In the present study, we have developed a 3D in vitro model of breast cancer bone metastasis mimicking later stages of breast cancer pathogenesis to the bones, using osteogenically differentiated human mesenchymal stem cells (MSCs) and human breast cancer cells (MDA-MB-231 & MCF-7) on nanoclay-based scaffolds. These two cell lines represent to highly and less metastatic breast cancer. Here we specifically describe the nature of bone regulation at metastatic site due to these two cell lines. Specifically, we have evaluated the effects of breast cancer cells secreted factors on bone formation in terms of the Wnt/ β-catenin signaling pathway. We noticed that stimulation of Wnt/β-catenin signaling in sequential cultures of MSCs with MCF-7 by endothelin-1 (ET-1) led to the increased bone formation while inhibition of Wnt/β-catenin signaling by dickkopf homolog 1 (DKK-1) exhibited reduced bone formation, thus mimicking mixed bone lesions observed in human breast cancer patients. We also evaluated drug-resistance of our 3D culture model using anti-cancer drug paclitaxel and found tumor-microenvironment released cytokine Interleukin-6 (IL-6) may be involved in conferring drug resistance via STAT3 pathway. Taken together, these critical findings not only further our understanding of breast cancer-induced bone metastasis but also have broader implications in patient-specific therapies in particular those that address skeletal complication due to metastasis.

Prostate cancer in its late stage is likely to metastasize to bone. A reliable marker for bone metastasis does not exist. This study evaluates the use of Raman mapping to evaluate subcellular changes and responses as a marker for bone metastasis. We have obtained patient tissues from prostate cancer at primary and secondary (bone sites). Raman spectra and Raman maps were obtained for these tissues. A complete and comprehensive study that involved comparison of the spectral maps from the human tissues was compared with data obtained from tumors grown using MDA PCa 2b prostate cell line grown on tissue engineered bone mimetic scaffolds. Our results indicate distinct differences and changes between the prostate cancer samples obtained at primary and secondary sites. Raman spectroscopic features in the lipid, protein, DNA regions of bone metastatic prostate cancer tissues represent very valuable diagnostic information since these experiments can be conducted in a clinical regime with minimal patient discomfort through use of fiber optics. We also used a variety of novel spectral and statistical methods such as PCA to classify the event of bone metasatsis. Overall, we demonstrate the potential for use of Raman Mapping conbined with powerful data analysis technologies for next generation metastasis diagnosis and prediction.

Two-dimensional materials, such as graphene and monolayer hexagonal Boron doped Graphene, are attractive for demonstrating fundamental physics in materials and potential applications in next-generation electronics. Atomic sheets containing hybridized bonds involving elements B and C over wide compositional ranges could result in new materials with properties complementary to those of graphene and Boron doped Graphene, enabling a rich variety of electronic structures with tunable properties and applications. Here we report the synthesis and characterization using spectroscopic techniques of large-area atomic layers of Boron doped Graphene material, consisting of hybridized, randomly distributed domains of B and C phases with compositions ranging from pure BNNTs to pure graphene. Our experimental and theoretical studies (based on Density Functional Theory and Continuum calculations) reveal that their structural features and bandgap are distinct from those of graphene and doped graphene. Boron -11 magic-angle spinning NMR experiments confirm the presence of boron in the sample, and the small quadrupolar broadening coupled with the center-of-gravity shift of -4.4 ppm is suggestive of a four-coordinate boron site. Similarly, the two-dimensional ¹¹B MQMAS NMR spectrum presents a broad signal centered at -4.4 ppm (Figure 1b). Slight spectral broadenings are consistent with a small distribution of local chemical environments, such as differences in the identity of geometry about the boron nearest neighbors in boron-doped graphene. This new form of hybrid Boron doped graphene material enables the development of bandgap-engineered applications in bioelectronics and biosensors and properties that are distinct from those of graphene and h-BN.

The structural and electronic properties of Fluorographene (FG) ultra-thin films directly grown on flexible and non-flexible insulating silicate substrates have been characterized. FG has been identified as an in-plane heterostructure consisting of sp² and sp³ hybridized carbon domains. ¹⁹F NMR shows a uniform distribution of fluorine throughout the plane in thin-film FG, unlike in other chemically derived FG variants, indicating the presence of sp³ carbon along with the sp² hybridized domains. It is found that dangling bonds (sp³ carbons without fluorine) cause paramagnetic behavior leading to sidebands in the NMR Spectra. The fluorine doping and band-structure of FG are further confirmed by spectroscopic ellipsometry studies, showing a band-gap of 1.2 eV, consistent with ab-initio SCAN-META GGA Functional based Density Functional Theory (DFT) calculations for 7% Fluorination of the Graphene honeycomb structure. This semiconducting FG layer has been identified as an ambipolar material from gate-controlled transport measurements. The transport properties of FG resemble those of reduced graphene oxide, another functionalized graphene derivative, with an Arrhenius type temperature dependence of resistance at high temperatures. This study unravels new avenues in atomic layer-based electronic circuitries and biosensors, where FG can be used as an active layer or dielectric support by tuning its fluorine content to the requisite level.

Cell agglomeration has been useful and crucial for tissue engineering, cell culturing and drug testing, such as for cancer drug development. And a 3D structured spheroid is superior to 2D cell cultures for many aspects: greater stability, longer life-span, more detailed cell-cell interactions, more accurate cell polarization depiction and so on. However, most of the methods require specific and complex experimental setups and the resulting agglomerates are generally slow or of poor quality. We propose a microfluidic device using surface acoustic waves (SAWs), a contact-free means, in the application of cell engineering. It is composed of a piezoelectric substrate, 127.86° Y-rotated X-propagating lithium niobate, and a focused interdigital transducer to generate focused surface waves with resonant frequency at 100 MHz. A conducting metal guiding layer is deposited on top of the substrate to overcome the beam steering and lateral diffraction problems due to the anisotropy nature of the piezoelectric material, further trapping the wave to a small region of a few wavelengths (~10¹ μm). The SAWs generated travel into a thin layer of liquid and diffract at the Rayleigh angle so that the acoustic radiation is coupled and propagates into the superstrate, which can be a bio-friendly container culturing cells. The localized acoustic streaming induced from the radiation carries and accumulates the cells to the center of the recirculation. By slightly tuning up the power, the stronger recirculation is able to levitate the periphery of the cell cluster and fold the structure to a three-dimensional configuration, and thus a spheroid can be formed. Our data shows the agglomerations can be created in less than 5 min, and the separation between the living human embryonic kidney cell agglomeration islands is as small as △x =720 μm. With the help of waveguides, the efficiency can be further improved by establishing multiple spheroid formations simultaneously in one well-free container. Therefore, this device is proven to be an effective, reliable and easy-to-handle approach for 3D cell manipulation and spheroid construction, rendering potentials in cell engineering.

First-generation nucleic acid analogs such as 2¢-deoxy-2′-fluoro-RNA (2′-F RNA), 2′-O-methyl RNA (2′-OMe RNA), and the phosphorothioates (PS-DNA/RNA) have remained staples of oligonucleotide modification strategies in the discovery and optimization of potential RNAi therapeutics.^1,2 The first FDA-approved siRNA drug, patisiran (Onpattro) contains eleven 2′-OMe ribonucleotides in the sense and antisense strands formulated in a lipid nanoparticle (LNP) delivery system.³ Despite their overall appeal in terms of enhanced nuclease resistance, increased pairing stability (2′-F/OMe RNAs) and safety, newer analogs are needed to further improve the therapeutic potential of unformulated siRNAs like GalNAc conjugates. The search for oligonucleotides with therapeutic efficacy has given rise to an expanding universe of analogs, including the second-generation 2'-O-(2-methoxyethyl)-RNA (MOE-RNA)⁴ and the third-generation bicyclic nucleic acids (BNAs or LNAs).⁵ The key enzyme in RNA interference (RNAi) is Argonaute 2 (Ago2), a dynamic multi-domain enzyme that binds multiple regions of the guide (antisense) and passenger (sense) siRNAs. Considering the complexity of protein-RNA interactions in RNA interference, it is imperative to expand the process of discovery and optimization of modified siRNAs to new analogs.⁶ We have explored the potential benefits for efficacy of incorporating into siRNAs sugar modifications like N-methylacetamide (NMA), 5′-E-vinylphosphonate (5′-E-VP) and, more recently, xeno-nucleic acid (XNA)⁷ residues such as glycol nucleic acid (GNA),⁸ altritol nucleic acid (ANA),⁹ and threofuranosyl nucleic acid (TNA).¹⁰ The talk will summarize siRNA modification strategies with an emphasis on regiospecific interactions between oligonucleotide and Ago2 and how structural insights based on crystallographic data for modified RNAs alone and in complex with Ago2 from molecular modeling studies can guide the choice of chemical modification at a given position of siRNA for optimizing its therapeutic efficacy.
1. Crooke, S. T.; Witztum, J. L.; Bennett, C. F.; Baker, B. F. Cell Metab. 2018, 27, 714-739.
2. Shen, X.; Corey, D. R. Nucleic Acids Res. 2018, 46, 1584-1600.
3. Sheridan, C. Nat. Biotechnol. 2017, 35, 995-997.
4. Teplova, M.; Minasov, G.; Tereshko, V.; Inamati, G.; Cook, P. D.; Manoharan, M.; Egli, M. Nat. Struct. Biol. 1999, 6, 535-539.
5. Pallan, P. S.; Allerson, C. R.; Berdeja, A.; Seth, P. P.; Swayze, E. E.; Prakash T. P.; Egli, M. Chem. Comm. 2012, 48, 8195-8197.
6. Egli, M.; Manoharan, M. Acc. Chem. Res. 2019, 52, 1036-1047.
7. Anosova, I.; Kowal, E. A.; Dunn, M. R.; Chaput, J. C.; van Horn, W. D.; Egli, M. Nucleic Acids Res. 2016, 44, 1007-1021.
8. Schlegel, M. K.; Foster, D. J.; Kel’in, A. V.; Zlatev, I.; Bisbe, A.; Jayaraman, M.; Lackey, J. G.; Rajeev, K. G.; Charisse, K.; Harp, J.; Pallan, P. S.; Maier, M. A.; Egli, M.; Manoharan, M. J. Am. Chem. Soc. 2017, 139, 8537-8546.
9. Ovaere, M.; Sponer, J.; Sponer, J. E.; Herdewijn, P.; Van Meervelt, L. Nucleic Acids Res. 2012, 40, 7573-7583.
10. Schöning, K.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.; Eschenmoser, A. Science 2000, 290, 1347-1351.

Cancer is a complex genetic moving target that requires a therapeutic modality capable of selectively targeting all driver oncogenes combined with the inherent ability to undergo pharmaco-evolution to target new mutations at pace with the cancer cell mutation rate. siRNA RNA-interference (RNAi) therapeutics fulfill these criteria and have great potential to treat cancer and other human diseases. However, due to their 14 kDa size and 40 negative anionic charges, siRNAs have no bioavailability to overcome a billion years of evolutionary defenses to prevent RNAs from crossing the endosomal lipid bilayer to enter the cytoplasm. Consequently, developing anti-cancer RNAi therapeutics requires targeted delivery to malignant cells and paradigm-shifting technology to enhance endosomal escape into the cytoplasm, the critical rate-limiting delivery step. Our research is focused on solving these problems by synthesis of bioreversible charge neutral RNAi prodrugs, called short interfering RiboNucleic Neutrals (siRNNs), and synthesis of novel hydrophilic-masked, hydrophobic endosomal escape domains (EEDs). For targeting domains, we have focused on conjugating antibodies to siRNN RNAi triggers to generate Antibody-RNAi Conjugates (ARCs).

Reduction sensitive linkers have the potential to transform the field of drug delivery due to their ease of use and selective cleavage in intracellular environments. However, despite their compelling attributes, using reduction sensitive linkers for biomolecule conjugation reactions is still challenging because linkers have not been developed that can efficiently modify aliphatic amines and release them rapidly and completely after reduction. Developing reduction sensitive self-immolative linkers for aliphatic amines has been challenging due to their poor leaving group ability and high pKa values. Here we present a traceless linker (TRAILER), which can for the first time modify aliphatic amines and release them rapidly and quantitatively after disulfide reduction. We show here that TRAILER can reversibly modify the lysine residues on the Cas9 protein, with the cell penetrating peptide Arg₁₀, and was able to generate a self delivering Cas9 RNP that could edit cells without transfection reagents. TRAILER has numerous applications in biotechnology given the ubiquitous presence of aliphatic amines on small molecule and protein therapeutics.

Non-coding ribonucleic acids (ncRNAs) are a diverse group of RNA molecules that are not translated into proteins following transcription. Mainly, microRNAs and long non-coding RNAs (lncRNAs) are predominant in this group. Aberrant expression of these ncRNAs has been observed in various pathological conditions including cancer. Especially, aberrant expression of microRNAs and the associated pathology has been extensively studied in cancer. Hence, targeting microRNAs has been considered a new generation of molecularly targeted therapy for various diseases. While downregulation of cellular microRNAs can be supplemented by delivering microRNA mimics, functional knock down of microRNAs that are overexpressed in diseases is a challenging task. The secondary structure of microRNAs determines how to functionally inhibit the translation of target mRNAs. MicroRNAs with even a few base homologies to the target can functionally regulate mRNA degradation/translation. The secondary structure of microRNA is crucial for recognizing target mRNA for functional effect. Since the double stranded RNA is an important intermediate in the biogenesis process of matured microRNA, delivering antisense microRNA for functional knock down of endogenous microRNAs needs a careful design. Especially challenging is their site-targeted delivery. We study antisense microRNAs with various structural determinants (mere homology, end loops, base pair modifications, artificial linkage, etc.,) by molecular dynamics of the duplex formation at 1 micro second and beyond, for the evaluation of the therapeutic effect of microRNAs in various cancer models (GBM: antisense miR-21 and antisense miR-100; Breast cancer antisense miR-21 and antisense miR-10b; Hepatocellular carcinoma: antisense miR-21, antisense miR-10b and antisense miR-100). In this presentation, we summarize how structural modification in antisense microRNAs can be used for potential applications in cancer therapy, either microRNA targeting as a monotherapy, or for improving clinical chemotherapy.

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Upon systemic administration of nanoparticles used in drug and gene delivery, they acquire a “biological identity” upon adsorption of plasma proteins. The protein corona layer has significant implication in the biodistribution, target specific localization, and toxicity of nanoparticle-based delivery systems. Additionally, specific enrichment of proteins may allow for an endogenous and more efficient approach for targeted delivery.
Using a wide ranging of lipid nanoparticle (LNP) formulation where individual lipid composition was varied, we have shown specific protein enrichment pattern and differential biodistribution and tumor-specific delivery of small interfering RNA (siRNA). For instance, DOTAP functionalized LNPs showed vitronectin enrichment and enhanced in vivo tumor delivery of the nanoparticles to αvβ3 expressed tumors and endothelial cells as compared to αvβ3 negative tumors.

Current advances in RNA-based technologies such as therapeutic gene editing, mRNA therapeutics, and vaccines have produced strong interest and attention in safe and effective nonviral vectors for nucleic acid delivery. Nevertheless, the comprehensive application of nucleic acid therapeutics is still limited by the need for better delivery systems. Thus, the analysis of these delivery materials can be advanced to offer sharper insights into SAR learnings and provide a clearer picture of their therapeutic potential. Lipid- and polymer-based materials can be regarded as the most prominent partners for nucleic acid delivery, providing in vivo transfection. Over the past few decades, a variety of cationic lipids and polymers have been synthesized, evaluated and applied as successful delivery vehicles for nucleic acids. Here, I will discuss some of the challenges for clinical translation of mRNA-based therapeutics and give examples of the biomaterials and delivery strategies we are investigating for the application of mRNA delivery.

We have recently developed a CRISPR powered, electronic biosensor termed, CRISPR Chip, capable of detecting the binding interactions between nuclease deactivated CRISPR with its target DNA sequence. CRISPR-Chip is a graphene field effect transistor (gFET) where the
surface of the graphene is functionalized with nuclease deactivated CRISPR RNA-guided ribonucleoproteins (dRNPs). dRNPs within the CRISPR-Chip construct interact with their target sequence by scanning the genomic sample until they find and bind to their target. The selective hybridization of the target DNA to the dRNP complex modulates the graphene’s conductivity and results in a detectable change in electrical signal output which can be measured with a simple handheld reader. CRISPR-Chip was able to detect dRNP binding to two commonly deleted exon target sequences within the human dystrophin gene, without amplification. CRISPR-chip is the first example of CRISPR-powered electronic transistors that harnesses the search function of CRISPR/Cas9 and the ultra-sensitivity of graphene-based nanoelectronics to enable a complete label-free and amplification-free DNA biosensor.