Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity.

For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.

Fabrication and use of silicon hollow-needle arrays to achieve tissue nanotransfection in mouse tissue in vivo.

Tissue nanotransfection (TNT) is an electromotive gene transfer technology that was developed to achieve tissue reprogramming in vivo. This protocol describes how to fabricate the required hardware, commonly referred to as a TNT chip, and use it for in vivo TNT. Silicon hollow-needle arrays for TNT applications are fabricated in a standardized and reproducible way. In <1 s, these silicon hollow-needle arrays can be used to deliver plasmids to a predetermined specific depth in murine skin in response to pulsed nanoporation. Tissue nanotransfection eliminates the need to use viral vectors, minimizing the risk of genomic integration or cell transformation. The TNT chip fabrication process typically takes 5-6 d, and in vivo TNT takes 30 min. This protocol does not require specific expertise beyond a clean room equipped for basic nanofabrication processes.

Rational design of on-chip gold plasmonic nanoparticles towards ctDNA screening.

This paper demonstrates the design, synthesis, simulation, and testing of three distinct geometries of plasmonic gold nanoparticles for on-chip DNA screening towards liquid biopsy. By employing a seed-mediated growth method, we have synthesized gold nanospheres, nanorods, and nanobipyramids. In parallel, we developed numerical simulations to understand the effects of nanoparticle geometry on the resonance features and refractive index sensitivity. Both experimental and simulation results were compared through a series of studies including in-solution and on-chip tests. We have thoroughly characterized the impact of nanoparticle geometry on the sensitivity to circulating tumor DNA, with immediate implications for liquid biopsy. The results agree well with theoretical predictions and simulations, including both bulk refractive index sensitivity and thin film sensitivity. Importantly, this work quantitatively establishes the link between nanoparticle geometry and efficacy in detecting rare circulating biomarkers. The nanobipyramids provided the highest sensitivity, approximately doubling the sensitivity compared to nanorods. To the best of our knowledge this is the first report carrying through geometric effects of simulation to clinically relevant biosensing. We put forth here synthesis and testing of three nanoparticle geometries, and a framework for both experimental and theoretical validation of plasmonic sensitivities towards liquid biopsy.

Single-Step Printable Hydrogel Microarray Integrating Long-Chain DNA for the Discriminative and Size-Specific Sensing of Nucleic Acids.

A simple approach to fabricating hydrogel-based DNA microarrays is reported by physically entrapping the rolling circle amplification (RCA) product inside printable in situ gelling hydrazone cross-linked poly(oligoethylene glycol methacrylate) hydrogels. The hydrogel-printed RCA microarray facilitates improved RCA immobilization (>65% even after vigorous washing) and resistance to denaturation relative to RCA-only printed microarrays in addition to size-discriminative sensing of DNA probes (herein, 27 or fewer nucleotides) depending on the internal porosity of the hydrogel. Furthermore, the high number of sequence repeats in the concatemeric RCA product enables high-sensitivity detection of complementary DNA probes without the need for signal amplification, with signal/noise ratios of 10 or more achieved over a short 30 min assay time followed by minimal washing. The inherent antifouling properties of the hydrogel enable discriminative hybridization in complex biological samples, particularly for short (∼10 nt) oligonucleotides whose hybridization in other assays tends to be transient and of low affinity. The scalable manufacturability and efficient performance of these hydrogel-printed RCA microarrays thus offer potential for rapid, parallel, and inexpensive sensing of short DNA/RNA biomarkers and ligands, a critical current challenge in diagnostic and affinity screening assays.

Developing a Portable Device for the Identification of miRNAs in Fluids.

In the present work we describe a novel system for the identification of microRNAs (miRNAs) in fluids. The method is based on combined novel 3D microarray technology using silk as scaffold and total internal reflection fluorescence microscopy (TIRFM), which allows for the rapid identification of miRNAs using a portable device.

Imputation for sequencing variants preselected to a customized low-density chip.

The sequencing variants preselected from association analyses and bioinformatics analyses could improve genomic prediction. In this study, the imputation of sequencing SNPs preselected from major dairy breeds in Denmark-Finland-Sweden (DFS) and France (FRA) was investigated for both contemporary animals and old bulls in Danish Jersey. For contemporary animals, a two-step imputation which first imputed to 54 K and then to 54 K + DFS + FRA SNPs achieved highest accuracy. Correlations between observed and imputed genotypes were 91.6% for DFS SNPs and 87.6% for FRA SNPs, while concordance rates were 96.6% for DFS SNPs and 93.5% for FRA SNPs. The SNPs with lower minor allele frequency (MAF) tended to have lower correlations but higher concordance rates. For old bulls, imputation for DFS and FRA SNPs were relatively accurate even for bulls without progenies (correlations higher than 97.2% and concordance rates higher than 98.4%). For contemporary animals, given limited imputation accuracy of preselected sequencing SNPs especially for SNPs with low MAF, it would be a good strategy to directly genotype preselected sequencing SNPs with a customized SNP chip. For old bulls, given high imputation accuracy for preselected sequencing SNPs with all MAF ranges, it would be unnecessary to re-genotype preselected sequencing SNPs.

Digital DNA microarray generation on glass substrates.

In this work we show how DNA microarrays can be produced batch wise on standard microscope slides in a fast, easy, reliable and cost-efficient way. Contrary to classical microarray generation, the microarrays are generated via digital solid phase PCR. We have developed a cavity-chip system made of a PDMS/aluminum composite which allows such a solid phase PCR in a scalable and easy to handle manner. For the proof of concept, a DNA pool composed of two different DNA species was used to show that digital PCR is possible in our chips. In addition, we demonstrate that DNA microarray generation can be realized with different laboratory equipment (slide cycler, manually in water baths and with an automated cartridge system). We generated multiple microarrays and analyzed over 13,000 different monoclonal DNA spots to show that there is no significant difference between the used equipment. To show the scalability of our system we also varied the size and number of the cavities located in the array region up to more than 30,000 cavities with a volume of less than 60 pL per cavity. With this method, we present a revolutionary tool for novel DNA microarrays. Together with new established label-free measurement systems, our technology has the potential to give DNA microarray applications a new boost.

Identifying multi-hit carcinogenic gene combinations: Scaling up a weighted set cover algorithm using compressed binary matrix representation on a GPU.

Despite decades of research, effective treatments for most cancers remain elusive. One reason is that different instances of cancer result from different combinations of multiple genetic mutations (hits). Therefore, treatments that may be effective in some cases are not effective in others. We previously developed an algorithm for identifying combinations of carcinogenic genes with mutations (multi-hit combinations), which could suggest a likely cause for individual instances of cancer. Most cancers are estimated to require three or more hits. However, the computational complexity of the algorithm scales exponentially with the number of hits, making it impractical for identifying combinations of more than two hits. To identify combinations of greater than two hits, we used a compressed binary matrix representation, and optimized the algorithm for parallel execution on an NVIDIA V100 graphics processing unit (GPU). With these enhancements, the optimized GPU implementation was on average an estimated 12,144 times faster than the original integer matrix based CPU implementation, for the 3-hit algorithm, allowing us to identify 3-hit combinations. The 3-hit combinations identified using a training set were able to differentiate between tumor and normal samples in a separate test set with 90% overall sensitivity and 93% overall specificity. We illustrate how the distribution of mutations in tumor and normal samples in the multi-hit gene combinations can suggest potential driver mutations for further investigation. With experimental validation, these combinations may provide insight into the etiology of cancer and a rational basis for targeted combination therapy.

Development of Gene Expression-Based Biomarkers on the nCounter® Platform for Immuno-Oncology Applications.

Biomarkers based on transcriptional profiling can be useful in the measurement of complex and/or dynamic physiological states where other profiling strategies such as genomic or proteomic characterization are not able to adequately measure the biology. One particular advantage of transcriptional biomarkers is the ease with which they can be measured in the clinical setting using robust platforms such as the NanoString nCounter system. The nCounter platform enables digital quantitation of multiplexed RNA from small amounts of blood, formalin-fixed, paraffin-embedded tumors, or other such biological samples that are readily available from patients, and the chapter uses it as the primary example for diagnostic assay development. However, development of diagnostic assays based on RNA biomarkers on any platform requires careful consideration of all aspects of the final clinical assay a priori, as well as design and execution of the development program in a way that will maximize likelihood of future success. This chapter introduces transcriptional biomarkers and provides an overview of the design and development process that will lead to a locked diagnostic assay that is ready for validation of clinical utility.

Modulated light-activated electrochemistry at silicon functionalized with metal-organic frameworks towards addressable DNA chips.

Modulated light-activated electrochemistry (MLAE) at semiconductor/liquid interfaces derived from light-addressable potentiometric sensor (LAPS) and light-activated electrochemistry (LAE) for addressable photoelectrochemical sensing has been proposed as a new sensor platform. In this system, a bias voltage is applied to create a depletion layer at the silicon/electrolyte interface. Meanwhile, intensity-modulated light illuminates the movable electrode to generate electron/hole pairs and causes a detectable local AC photocurrent. The AC measurement showed a higher signal-to-noise ratio (SNR) of photocurrents compared to the traditional DC response, while a steeper photocurrent-voltage (I-V) curve than that of LAPS with an insulating layer was obtained. Furthermore, to stabilize and functionalize the silicon substrate, metal-organic framework (MOF) nanoparticles were grown in-situ on the silicon electrode. The successful modification was validated by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The AC photocurrent increased as a result of the adsorption of negatively charged DNA, which contributed to the enhancement of the cathodic reduction process at the semiconductor electrodes, indicating a different response mechanism of MLAE from LAPS. The results obtained demonstrate the potential of MOF functionalized MLAE as a robust platform for light-addressable DNA chips with high sensitivity and specificity.

Hybridization to Homemade Microarrays.

Competitive hybridization of labeled nucleic acids to a microarray is conceptually similar to other hybridization methods, such as Southern blotting. For massively multiplexed microarrays, the adoption of two-color hybridization schemes has been a significant advance. The use of two colors-typically Cy3- and Cy5-labeled nucleic acids-makes it possible to control for factors that affect hybridization intensity, including the number of labeled nucleotides and the T m of each oligonucleotide. Thus, the difference in intensity among spots on a microarray can be quantified and analyzed to assess biological phenomena, like changes in gene expression or details of transcript structure. This protocol for hybridization is conceptually straightforward-"cold" (nonfluorescent) blocking nucleotide is added to the mixed nucleic acid material, Hybridization buffer is added, and the mixture is applied to the microarray surface. Hybridization occurs overnight, after which the microarray is washed and scanned.

How to copy and paste DNA microarrays.

Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of the copied DNA microarray can also be altered compared to the original by producing copies of different resolutions. As a homage to the very first photocopy made by Chester Charlson and Otto Kornei, we performed a lookalike DNA microarray copy exactly 80 years later. Those copies were also used for label-free real-time kinetic binding assays of apo-dCas9 to double stranded DNA and of thrombin to single stranded DNA. Since each DNA microarray copy was made with only 5 µl of spPCR mix, the whole process is cost-efficient. Hence, our DNA microarray copier has a great potential for becoming a standard lab tool.

MicroRNA detection on microsensor arrays by SPR imaging measurements with enzymatic signal enhancement.

We investigated sequence-specific and simultaneous microRNA (miRNA) detections by surface plasmon resonance (SPR) imaging measurements on SPR chips possessing an Au spot array modified with probe DNAs based on a miRNA-detection-selective SPR signal amplification method. MiRNAs were detected with the detection limit of the attomole level by SPR imaging measurements for different miRNA concentrations on a single chip. SPR signals were enhanced based on a combination process of sequence-specific hybridization of the miRNA to the probe DNAs, extension reaction of polyadenine (poly(A)) tails by poly(A) polymerase, binding of a ternary complex of T30-biotin/horseradish peroxidase (HRP)-biotin/streptavidin to the poly(A) tails, and the oxidation reaction of tetramethylbenzidine (TMB) on the HRP by providing a blue precipitate on the surface. This process sequence-specifically and dramatically amplified the SPR signals. This is a simple, cost-effective, and feasible signal amplification method based on the organic compound TMB instead of metal nanoparticles.

Integration of rolling circle amplification and optomagnetic detection on a polymer chip.

Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis.

Mosquito-Borne Viruses and Insect-Specific Viruses Revealed in Field-Collected Mosquitoes by a Monitoring Tool Adapted from a Microbial Detection Array.

Several mosquito-borne diseases affecting humans are emerging or reemerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. In this study, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance biosurveillance by detecting microbes present in Aedes aegypti, Aedes albopictus, and Culex mosquitoes, which are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA were tested in mosquito samples spiked with different concentrations of dengue virus (DENV), revealing a detection limit of >100 but <1,000 PFU/ml. Additionally, field-collected mosquitoes from Chicago, IL, and College Station, TX, of known infection status (West Nile virus [WNV] and Culex flavivirus [CxFLAV] positive) were tested on the LLMDA to confirm its efficiency. Mosquito field samples of unknown infection status, collected in San Antonio, TX, and the Lower Rio Grande Valley (LRGV), TX, were run on the LLMDA and further confirmed by PCR or quantitative PCR (qPCR). The analysis of the field samples with the LLMDA revealed the presence of cell-fusing agent virus (CFAV) in A. aegypti populations. Wolbachia was also detected in several of the field samples (A. albopictus and Culex spp.) by the LLMDA. Our findings demonstrated that the LLMDA can be used to detect multiple arboviruses of public health importance, including viruses that belong to the Flavivirus, Alphavirus, and Orthobunyavirus genera. Additionally, insect-specific viruses and bacteria were also detected in field-collected mosquitoes. Another strength of this array is its ability to detect multiple viruses in the same mosquito pool, allowing for the detection of cocirculating pathogens in an area and the identification of potential ecological associations between different viruses. This array can aid in the biosurveillance of mosquito-borne viruses circulating in specific geographical areas.IMPORTANCE Viruses associated with mosquitoes have made a large impact on public and veterinary health. In the United States, several viruses, including WNV, DENV, and chikungunya virus (CHIKV), are responsible for human disease. From 2015 to 2018, imported Zika cases were reported in the United States, and in 2016 to 2017, local Zika transmission occurred in the states of Texas and Florida. With globalization and a changing climate, the frequency of outbreaks linked to arboviruses will increase, revealing a need to better detect viruses in vector populations. With the capacity of the LLMDA to detect viruses, bacteria, and fungi, this study highlights its ability to broadly screen field-collected mosquitoes and contribute to the surveillance and management of arboviral diseases.

Coaxial sensing bio-amplifier for ultrasensitive detections of circulating tumor DNAs.

We herein report the first attempt to engineer a coaxial-sensing 3D amplifier able to achieve dynamic self-assembly in response to a mutated-ctDNA target. A bio-nanofiber is firstly manufactured via an ingenious double-channel electrostatic spinning and DNA rolling circle replication (RCR) technology, which offered an ideal scaffold for assembly of 3D amplifier activated by target recognition. The coaxial-controllable signal amplifier presented several advantages. (1) Given its "coaxial sensing effect", the proposed bio-amplifier played the coaxial transduction for signal enrichment to vastly increase sensitivity, capable of discriminating a single-base mismatched sequence from the perfectly complementary one, using ctDNA-134A as a model analyte. (2) Due to "covalent bridges lock effect" in an identifying chip with locked nucleic acid beacons, this 3D amplifier expressed high specificity and biostability toward seven different mutated-ctDNAs. (3) Profiting from special configuration of bioactive nanofibers and DNA replication programming, this catalytic bio-amplifier possessed "signal enrichment effect", which enhanced dynamic range toward ctDNA-134A detection and hybridized without any external indicators. This innovative bio-amplifier has a detection limit of 5.1 aM for ctDNA-134A with superior specificity, excellent sensitivity, and good performance. This pioneered method was further applied for broadly differentiate cells and evaluate changes in the expression levels of intracellular mutated-ctDNAs.

Automated DNA hybridization transfer with movable super-paramagnetic microbeads in a microflow reactor.

An automated DNA hybridization transfer in a microflow reactor is demonstrated by moving paramagnetic beads between two spatially separate solutions with different pH values. The microbeads-based microfluidic platform is fully automated and programmable. It employs a robust chemical procedure for specific DNA hybridization transfer in microfluidic devices under isothermal conditions based on reversible pH alterations. The method takes advantage of high-speed DNA hybridization and denaturation on beads under flow conditions, high fidelity of DNA hybridization, and small sample volumes. The microfluidic platform presented is saleable and applicable to many areas of modern biotechnology such as DNA hybridization chip microarrays, molecular computation, on-chip selection of functional nucleic acids, high-throughput screening of chemical libraries for drug discovery, and DNA amplification and sequencing.

Evaluating DNA methylation age on the Illumina MethylationEPIC Bead Chip.

DNA methylation age (DNAm age) has become a widely utilized epigenetic biomarker for the aging process. The Horvath method for determining DNAm age is perhaps the most widely utilized and validated DNA methylation age assessment measure. Horvath DNAm age is calculated based on methylation measurements at 353 loci, present on Illumina's 450k and 27k DNA methylation microarrays. With increasing use of the more recently developed Illumina MethylationEPIC (850k) microarray, it is worth revisiting this aging measure to evaluate estimation differences due to array design. Of the requisite 353 loci, 17 are missing from the 850k microarray. Similarly, an alternate, 71 loci DNA methylation age assessment measure created by Hannum et al. is missing 6 requisite loci. Using 17 datasets with 27k, 450k, and/or 850k methylation data, we compared each sample's epigenetic age estimated from all 353 loci required by the Horvath DNAm age calculator, and using only the 336 loci available on the 850k array. In 450k/27k data, removing loci not on the 850k array resulted in underestimation of Horvath's DNAm age. Underestimation of Horvath DNAm age increased from ages 0 to ~20, remaining stable thereafter (mean deviation = -3.46 y, SD = 1.13 for individuals ≥20 years). Underestimation of Horvath's DNAm age by the reduced 450k/27k data was similar to the underestimation observed in the 850k data indicating it is driven by missing probes. In analogous examination of Hannum's DNAm age, the magnitude and direction of epigenetic age misestimation varied with chronological age. In conclusion, inter-array deviations in DNAm age estimations may be largely driven by missing probes between arrays, despite default probe imputation procedures. Though correlations and associations based on Horvath's DNAm age may be unaffected, researchers should exercise caution when interpreting results based on absolute differences in DNAm age or when mixing samples assayed on different arrays.

Battery Powered Portable Thermal Cycler for Continuous-Flow Polymerase Chain Reaction Diagnosis by Single Thermostatic Thermoelectric Cooler and Open-Loop Controller.

Temperature control is the most important and fundamental part of a polymerase chain reaction (PCR). To date, there have been several methods to realize the periodic heating and cooling of the thermal-cycler system for continuous-flow PCR reactions, and three of them were widely used: the thermo-cycled thermoelectric cooler (TEC), the heating block, and the thermostatic heater. In the present study, a new approach called open-loop controlled single thermostatic TEC was introduced to control the thermal cycle during the amplification process. Differing from the former three methods, the size of this microdevice is much smaller, especially when compared to the microdevice used in the heating block method. Furthermore, the rising and cooling speed of this method is much rapider than that in a traditional TEC cycler, and is nearly 20-30% faster than a single thermostatic heater. Thus, a portable PCR system was made without any external heat source, and only a Teflon tube-wrapped TEC chip was used to achieve the continuous-flow PCR reactions. This provides an efficient way to reduce the size of the system and simplify it. In addition, through further experiments, the microdevice is not only found to be capable of amplification of a PCR product from Human papillomavirus type 49 (Genbank ref: X74480.1) and Rubella virus (RUBV), but also enables clinical diagnostics, such as a test for hepatitis B virus.

Large-Scale, Quantitative Protein Assays on a High-Throughput DNA Sequencing Chip.

High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56 × 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.