Comparing solution-gate and bottom-gate nanowire field-effect transistors on pH sensing with different salt concentrations and surface modifications.

Field-effect transistors (FETs) have been developed as pH sensors by using various device structures, fabrication technologies, and sensing film materials. Different transistor structures, like extended-gate (EG) FETs, floating-gate FET sensors, and dual-gate (DG) FETs, can enhance the sensor performance. In this article, we report the effects of using solution-gate and bottom-gate FET configurations on pH sensing and investigate the influence of different ionic concentrations of buffers in the measured signals. The surface charge of hafnium dioxide (HfO2) affected by the buffer pH, with/without the modification of polyethylene glycol (PEG) terminated with hydroxyl groups, and the location of applied gate voltage are vital factors to the sensor performance in pH sensing. Based on the results, the solution-gate FET exhibits good pH sensitivity even in the high ionic strength solutions of bis-tris propane (BTP), and these values of pH sensitivity are close to the Nernst limit (59.2 mV/pH). In general, silane-PEG-OH modification can reduce the deviations of measured signals in pH sensing. The performance of bottom-gate FET is inferior in the BTP buffers with high ionic solutions but suitable to be operated in low ionic concentrations, such as 0.1, 1, and 10 mM BTP buffers. The size of the ions was also studied and discussed. The solution-gate FET demonstrates excellent performance under high ionic strengths, meaning a more significant potential for detecting biological molecules under physiological conditions.

PLBD: protein-ligand binding database of thermodynamic and kinetic intrinsic parameters.

We introduce a protein-ligand binding database (PLBD) that presents thermodynamic and kinetic data of reversible protein interactions with small molecule compounds. The manually curated binding data are linked to protein-ligand crystal structures, enabling structure-thermodynamics correlations to be determined. The database contains over 5500 binding datasets of 556 sulfonamide compound interactions with the 12 catalytically active human carbonic anhydrase isozymes defined by fluorescent thermal shift assay, isothermal titration calorimetry, inhibition of enzymatic activity and surface plasmon resonance. In the PLBD, the intrinsic thermodynamic parameters of interactions are provided, which account for the binding-linked protonation reactions. In addition to the protein-ligand binding affinities, the database provides calorimetrically measured binding enthalpies, providing additional mechanistic understanding. The PLBD can be applied to investigations of protein-ligand recognition and could be integrated into small molecule drug design. Database URL https://plbd.org/.

Paper/PMMA hybrid device with a microvalve-controlled design for exosome isolation and analysis.

This study proposes a paper/PMMA hybrid device designed to isolate exosomes and extract exosomal miRNA, followed by quantitative analysis. It aims to provide simplified and convenient sample preparation for potential point-of-care testing (POCT) processes. In contrast to previous work conducted by our research team, which focused on isolating exosomes and exosomal nucleic acids, this study introduces a novel approach by integrating paper and a PMMA mold with a microvalve controlled design. This innovative method enables the entire process to be performed on paper. The pressure on the paper could be adjusted by turning the screw upon the valve to change the pore size and permeability of the paper, which achieved the effect of controlling the flow rate of fluids. The paper was designed to have an immunoaffinity area for capturing exosomes and a sol-gel silica coating area for extracting miRNA. The paper-based ELISA (p-ELISA) exhibited a limit of detection and a limit of quantitation of 6 × 107 and 5.4 × 108 particles/mL, respectively, for exosome measurement. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed that the Ct (threshold cycle) value for quantifying the miR-21 in the miRNAs extracted by the proposed paper/PMMA hybrid device was comparable to the Ct value of the commercial extraction kit. The developed paper/PMMA hybrid device with a microvalve-controlled design should be incorporated into the POCT system to extract exosomal miRNAs.

Paper-Based Exosomal MicroRNA-21 Detection for Wound Monitoring: A Proof of Concept and Clinical Validation Trial Study.

Emerging evidence has shown that microRNAs play pivotal roles in wound healing. MicroRNA-21 (miR-21) was previously found to upregulate in order to fulfill an anti-inflammation role for wounds. Exosomal miRNAs have been identified and explored as essential markers for diagnostic medicine. However, the role of exosomal miR-21 in wounds has yet to be well studied. In order to facilitate the early management of poorly healing wounds, we developed an easy-to-use, rapid, paper-based microfluidic-exosomal miR-21 extraction device to determine wound prognosis in a timely manner. We isolated and then quantitatively examined exosomal miR-21 in wound fluids from normal tissues and acute and chronic wounds. Eight improving wounds displayed lower levels of exosomal miR-21 expression after wound debridement. However, four instances of increased exosomal miR-21 expression levels were notably associated with patients with poor healing wounds despite aggressive wound debridement, indicating a predictive role of tissue exosomal miR-21 for wound outcome. Paper-based nucleic acid extraction device provides a rapid and user-friendly approach for evaluating exosomal miR-21 in wound fluids as a means of monitoring wounds. Our data suggest that tissue exosomal miR-21 is a reliable marker for determining current wound status.

Molecular effects of site-specific phosphate-methylated primer on the structure and motions of Taq DNA polymerase.

Polymerase chain reaction (PCR) is a powerful molecular biology assay for gene detection and quantification. Conventional DNA primers for PCR often suffer from poor sensitivity in specific gene detection. Recently, oligonucleotides containing methyl phosphotriester (MPTE-DNA) have been developed with enhanced DNA hybridization and improved gene detection sensitivity. Yet, site-specific MPTE-modifications on DNA primers have been reported to affect PCR amplification efficiencies while the detailed mechanism remains elusive. Here, we utilized molecular dynamics (MD) simulation to examine the effects of site-specific MPTE-modified primers on the structure and motions of DNA/Taq polymerase complexes. All tested MPTE-DNA/Taq complexes exhibited RMSD values below 2 Å, indicating insignificant effects of all methylation sites on the complex stability. The energetic and hydrogen-bonding analyses suggest minor effects of methylation at t-3, t-4, t-6, and t-7 positions on the DNA-Taq interaction. Principal component analyses further reveal that only t-3, and t-7 methylations preserve the motions of the Taq thumb domain. The site-specific methylation affects the interaction between DNA and the surrounding protein residues, resulting in allosteric-like effects on the DNA/Taq complex. The MD data complement the best experimentally observed PCR efficacies for the t-3 and t-7 positions among all tested MPTE-primers. The unveiled molecular insights can benefit the design of novel PCR primers for improving genetic testing platforms.

Ultrasensitive Detection of Interleukin 6 by Using Silicon Nanowire Field-Effect Transistors.

Interleukin 6 (IL-6) has been regarded as a biomarker that can be applied as a predictor for the severity of COVID-19-infected patients. The IL-6 level also correlates well with respiratory dysfunction and mortality risk. In this work, three silanization approaches and two types of biorecognition elements were used on the silicon nanowire field-effect transistors (SiNW-FETs) to investigate and compare the sensing performance on the detection of IL-6. Experimental data revealed that the mixed-SAMs-modified silica surface could have superior surface morphology to APTES-modified and APS-modified silica surfaces. According to the data on detecting various concentrations of IL-6, the detection range of the aptamer-functionalized SiNW-FET was broader than that of the antibody-functionalized SiNW-FET. In addition, the lowest concentration of valid detection for the aptamer-functionalized SiNW-FET was 2.1 pg/mL, two orders of magnitude lower than the antibody-functionalized SiNW-FET. The detection range of the aptamer-functionalized SiNW-FET covered the concentration of IL-6, which could be used to predict fatal outcomes of COVID-19. The detection results in the buffer showed that the anti-IL-6 aptamer could produce better detection results on the SiNW-FETs, indicating its great opportunity in applications for sensing clinical samples.

In-Silico Selection of Aptamer Targeting SARS-CoV-2 Spike Protein.

Aptamers are single-stranded, short DNA or RNA oligonucleotides that can specifically bind to various target molecules. To diagnose the infected cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in time, numerous conventional methods are applied for viral detection via the amplification and quantification of DNA or antibodies specific to antigens on the virus. Herein, we generated a large number of mutated aptamer sequences, derived from a known sequence of receptor-binding domain (RBD)-1C aptamer, specific to the RBD of SARS-CoV-2 spike protein (S protein). Structural similarity, molecular docking, and molecular dynamics (MD) were utilized to screen aptamers and characterize the detailed interactions between the selected aptamers and the S protein. We identified two mutated aptamers, namely, RBD-1CM1 and RBD-1CM2, which presented better docking results against the S protein compared with the RBD-1C aptamer. Through the MD simulation, we further confirmed that the RBD-1CM1 aptamer can form the most stable complex with the S protein based on the number of hydrogen bonds formed between the two biomolecules. Based on the experimental data of quartz crystal microbalance (QCM), the RBD-1CM1 aptamer could produce larger signals in mass change and exhibit an improved binding affinity to the S protein. Therefore, the RBD-1CM1 aptamer, which was selected from 1431 mutants, was the best potential candidate for the detection of SARS-CoV-2. The RBD-1CM1 aptamer can be an alternative biological element for the development of SARS-CoV-2 diagnostic testing.

Paper-Based Devices for Capturing Exosomes and Exosomal Nucleic Acids From Biological Samples.

Exosomes, nanovesicles derived from cells, contain a variety of biomolecules that can be considered biomarkers for disease diagnosis, including microRNAs (miRNAs). Given knowledge and demand, inexpensive, robust, and easy-to-use tools that are compatible with downstream nucleic acid detection should be developed to replace traditional methodologies for point-of-care testing (POCT) applications. This study deploys a paper-based extraction kit for exosome and exosomal miRNA analytical system with some quantifying methods to serve as an easy sample preparation for a possible POCT process. Exosomes concentrated from HCT116 cell cultures were arrested on paper-based immunoaffinity devices, which were produced by immobilizing anti-CD63 antibodies on Whatman filter paper, before being subjected to paper-based silica devices for nucleic acids to be trapped by silica nanoparticles adsorbed onto Whatman filter paper. Concentrations of captured exosomes were quantified by enzyme-linked immunosorbent assay (ELISA), demonstrating that paper-based immunoaffinity devices succeeded in capturing and determining exosome levels from cells cultured in both neutral and acidic microenvironments, whereas microRNA 21 (miR-21), a biomarker for various types of cancers and among the nucleic acids absorbed onto the silica devices, was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) to prove that paper-based silica devices were capable of trapping exosomal nucleic acids. The developed paper-based kit and the devised procedure was successfully exploited to isolate exosomes and exosomal nucleic acids from different biological samples (platelet-poor plasma and lesion fluid) as clinical applications.

Optimizing surface modification of silicon nanowire field-effect transistors by polyethylene glycol for MicroRNA detection.

MicroRNA (miRNA) sensing plays an essential role in the diagnosis of several diseases, especially cancers, for appropriate intervention and treatment. However, quantifying miRNA demands highly sensitive and selective assays which can distinguish analogous sequences with low abundance in bio-samples and determine wide range of concentrations. In this report, we present a novel technique satisfying all those requirements by modifying silicon nanowire field-effect transistors (SiNWFETs) with 2-component mixed self-assembled monolayers (mSAMs) of polyethylene glycol (PEG) at different ratios (silane-PEG-NH2:silane-PEG-OH = 1:1, 1:3, and 1:5) and glutaraldehyde to immobilize DNA probes for miRNA-21 detection, a biomarker in several types of cancers. Empirical results reveal that all the fabricated PEG-SiNWFET DNA biosensors could quantify miRNA-21 within 1 fM - 10 pM. Especially, the ones modified with silane-PEG-NH2:silane-PEG-OH = 1:3 exhibited an outstanding performance to recognize miRNA-21 at an ultra-low concentration of 10 aM in the dynamic range up to 6 orders of magnitude (10 aM - 10 pM). This approach is more convenient, analytical competitive, and cost-effective in comparison with currently used methods for nucleic acid testing because of label- and amplification-free characteristics. It is therefore not only feasible for miRNA detection by SiNWFET-based biosensors but also potential for clinical applications of disease diagnosis with oligonucleotide biomarkers.

Phosphate-Methylated Oligonucleotides as a Novel Primer for PCR and RT-PCR.

This chapter introduces neutralized DNA (nDNA) as a novel design for the primers of PCR and RT-PCR by methylating phosphate groups of some oligonucleotides in their structures. It starts with an introduction of the nDNA which possesses an electrically chimeric neutral backbone as well as the proposed standards in designing nDNA as a novel primer for PCR and RT-PCR , concluded from various experimental results presented afterward. The primary content comprises empirical data from PCR to compare nDNA and unmodified DNA as primers in terms of ability to distinguish and amplify mismatch templates, activities of polymerase enzymes, melting temperature of double-stranded sequences, and the trials and discussions on various modified positions of the nDNA primers. In summary, nDNA exhibited outstanding performance as a primer for PCR and RT-PCR , compared to unmodified DNA, and is expected to be expanded in diverse applications which require enhanced specificity.

Switching the Inhibitor-Enzyme Recognition Profile via Chimeric Carbonic Anhydrase XII.

A key part of the optimization of small molecules in pharmaceutical inhibitor development is to vary the molecular design to enhance complementarity of chemical features of the compound with the positioning of amino acids in the active site of a target enzyme. Typically this involves iterations of synthesis, to modify the compound, and biophysical assay, to assess the outcomes. Selective targeting of the anti-cancer carbonic anhydrase isoform XII (CA XII), this process is challenging because the overall fold is very similar across the twelve CA isoforms. To enhance drug development for CA XII we used a reverse engineering approach where mutation of the key six amino acids in the active site of human CA XII into the CA II isoform was performed to provide a protein chimera (chCA XII) which is amenable to structure-based compound optimization. Through determination of structural detail and affinity measurement of the interaction with over 60 compounds we observed that the compounds that bound CA XII more strongly than CA II, switched their preference and bound more strongly to the engineered chimera, chCA XII, based on CA II, but containing the 6 key amino acids from CA XII, behaved as CA XII in its compound recognition profile. The structures of the compounds in the chimeric active site also resembled those determined for complexes with CA XII, hence validating this protein engineering approach in the development of new inhibitors.

Uncertainty in protein-ligand binding constants: asymmetric confidence intervals versus standard errors.

Equilibrium binding constants (Kb) between chemical compounds and target proteins or between interacting proteins provide a quantitative understanding of biological interaction mechanisms. Reported uncertainties of measured experimental parameters are critical for decision-making in many scientific areas, e.g., in lead compound discovery processes and in comparing computational predictions with experimental results. Uncertainties in measured Kb values are commonly represented by a symmetric normal distribution, often quoted in terms of the experimental value plus-minus the standard deviation. However, in general, the distributions of measured Kb (and equivalent Kd) values and the corresponding free energy change ΔGb are all asymmetric to varying degree. Here, using a simulation approach, we illustrate the effect of asymmetric Kb distributions within the realm of isothermal titration calorimetry (ITC) experiments. Further we illustrate the known, but perhaps not widely appreciated, fact that when distributions of any of Kb, Kd and ΔGb are transformed into each other, their degree of asymmetry is changed. Consequently, we recommend that a more accurate way of expressing the uncertainties of Kb, Kd, and ΔGb values is to consistently report 95% confidence intervals, in line with other authors' suggestions. The ways to obtain such error ranges are discussed in detail and exemplified for a binding reaction obtained by ITC.

Combination of Aptamer Amplifier and Antigen-Binding Fragment Probe as a Novel Strategy to Improve Detection Limit of Silicon Nanowire Field-Effect Transistor Immunosensors.

Detecting proteins at low concentrations in high-ionic-strength conditions by silicon nanowire field-effect transistors (SiNWFETs) is severely hindered due to the weakened signal, primarily caused by screening effects. In this study, aptamer as a signal amplifier, which has already been reported by our group, is integrated into SiNWFET immunosensors employing antigen-binding fragments (Fab) as the receptors to improve its detection limit for the first time. The Fab-SiNWFET immunosensors were developed by immobilizing Fab onto Si surfaces modified with either 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) (Fab/APTES-SiNWFETs), or mixed self-assembled monolayers (mSAMs) of polyethylene glycol (PEG) and GA (Fab/PEG-SiNWFETs), to detect the rabbit IgG at different concentrations in a high-ionic-strength environment (150 mM Bis-Tris Propane) followed by incubation with R18, an aptamer which can specifically target rabbit IgG, for signal enhancement. Empirical results revealed that the signal produced by the sensors with Fab probes was greatly enhanced compared to the ones with whole antibody (Wab) after detecting similar concentrations of rabbit IgG. The Fab/PEG-SiNWFET immunosensors exhibited an especially improved limit of detection to determine the IgG level down to 1 pg/mL, which has not been achieved by the Wab/PEG-SiNWFET immunosensors.

Improve sample preparation process for miRNA isolation from the culture cells by using silica fiber membrane.

In clinical applications of miRNAs, the purity and quality of the testing samples are very critical, especially the obtained tissue sample volume is limited. If the extracted miRNAs are contaminated or different in quality before analysis, it will increase the variance of the analysis result and make the medical information judgment incorrect and cannot be portable. Herein, we improved the commercially extraction kit by realizing the fundamental mechanism and hoped to serve finding optimal procedures for increasing the recovery of miRNAs extracted from cultured cells. In the adsorption process, the factors, like increasing the ethanol concentration or adding Ca2+, could influence the RNA adsorption were investigated. For the elution process, the effect caused by raising the elution temperature and raising the pH value of elution buffer was studied. Finally, the conditions for miRNA extraction are optimal modified by using a 65% (v/v) solution of ethanol in the adsorption process, and using TE buffer with the pH value of 8.0 and raising the temperature to 55 °C in the elution. According to the quantified results, the improved extraction kit can promote the recovery of endogenous miR-21 by about 6 times by using the optimal extraction conditions comparing with the miRNeasy Mini Kit.

Predicting Future Prospects of Aptamers in Field-Effect Transistor Biosensors.

Aptamers, in sensing technology, are famous for their role as receptors in versatile applications due to their high specificity and selectivity to a wide range of targets including proteins, small molecules, oligonucleotides, metal ions, viruses, and cells. The outburst of field-effect transistors provides a label-free detection and ultra-sensitive technique with significantly improved results in terms of detection of substances. However, their combination in this field is challenged by several factors. Recent advances in the discovery of aptamers and studies of Field-Effect Transistor (FET) aptasensors overcome these limitations and potentially expand the dominance of aptamers in the biosensor market.

Field-Effect Transistor Biosensors for Biomedical Applications: Recent Advances and Future Prospects.

During recent years, field-effect transistor biosensors (Bio-FET) for biomedical applications have experienced a robust development with evolutions in FET characteristics as well as modification of bio-receptor structures. This review initially provides contemplation on this progress by briefly summarizing remarkable studies on two aforementioned aspects. The former includes fabricating unprecedented nanostructures and employing novel materials for FET transducers whereas the latter primarily synthesizes compact molecules as bio-probes (antibody fragments and aptamers). Afterwards, a future perspective on research of FET-biosensors is also predicted depending on current situations as well as its great demand in clinical trials of disease diagnosis. From these points of view, FET-biosensors with infinite advantages are expected to continuously advance as one of the most promising tools for biomedical applications.

Signal Enhancement of Silicon Nanowire Field-Effect Transistor Immunosensors by RNA Aptamer.

Silicon nanowire field-effect transistors (SiNW-FETs) have been demonstrated as a highly sensitive platform for label-free detection of a variety of biological and chemical entities. However, detecting signal from immunoassays by nano-FETs is severely hindered by the distribution of different charged groups of targeted entities, their binding orientation, and distances to the surface of the FET. Aptamers have been widely applied as a recognition element for plentiful biosensors because of small molecular sizes and moderate to high specific binding affinity with different types of molecules. In this study, we propose an effective approach to enhance the electrical responses of both direct (6×-histidine) and sandwich (amyloid ß 1-42) immunoassays in SiNW-FETs with R18, a highly negative charged RNA aptamer against rabbit immunoglobulin G (IgG). Empirical results presented that the immunosensors targeted with R18 expressed a significantly stabilized and amplified signal compared to the ones without this aptamer. The research outcome provides applicability of the highly negative charged aptamer as a bioamplifier for immunoassays by FETs.

Neutralized chimeric DNA probe for the improvement of GC-rich RNA detection specificity on the nanowire field-effect transistor.

Silicon nanowire (SiNW) field-effect transistors (FETs) is a powerful tool in genetic molecule analysis because of their high sensitivity, short detection time, and label-free detection. In nucleic acid detection, GC-rich nucleic acid sequences form self- and cross-dimers and stem-loop structures, which can easily obtain data containing signals from nonspecific DNA binding. The features of GC-rich nucleic acid sequences cause inaccuracies in nucleic acid detection and hinder the development of precision medicine. To improve the inaccurate detection results, we used phosphate-methylated (neutral) nucleotides to synthesize the neutralized chimeric DNA oligomer probe. The probe fragment originated from a primer for the detection of hepatitis C virus (HCV) genotype 3b, and single-mismatched and perfect-matched targets were designed for single nucleotide polymorphisms (SNP) detection on the SiNW FET device. Experimental results revealed that the HCV-3b chimeric neutralized DNA (nDNA) probe exhibited better performance for SNP discrimination in 10 mM bis-tris propane buffer at 25 °C than a regular DNA probe. The SNP discrimination of the nDNA probe could be further improved at 40 °C on the FET device. Consequently, the neutralized chimeric DNA probe could successfully distinguish SNP in the detection of GC-rich target sequences under optimal operating conditions on the SiNW FET device.

Designed phosphate-methylated oligonucleotides as PCR primers for SNP discrimination.

Polymerase chain reaction (PCR) is a powerful technique for the detection and quantification of nucleic acids and has enormous applications to research in molecular biology. Certain inherited diseases, caused by single nucleotide mutations, however, are difficult to identify by PCR, using DNA primers and probes, in a situation where a false diagnosis may lead to incorrect or delayed treatment. With the aim of enhancing the specificity of PCR, we used novel chemically synthesized oligonucleotides containing site-specific methyl phosphotriester (MPTE) inter-nucleoside linkage(s) as primers and probes. The methyl phosphotriester linkages carry no charge, so the reduction in the electrostatic repulsion of an MPTE-DNA/DNA duplex shows stronger hybridization affinity compared to a DNA/DNA duplex. However, the electrosteric effects introduced by the methyl group may result in instability of the double-stranded DNA (dsDNA) formed. With the use of specific MPTE modification sites and optimization of the number of MPTE modifications, greater delta melting temperature (ΔTm) may be obtained, in concert with adjustment of PCR operating conditions, especially with respect to the annealing temperature, to achieve more discriminatory results between the target template and the perfectly matched primer and the mismatched primer. In single nucleotide polymorphism (SNP) genotyping, the results demonstrated that MPTE-modified probes can improve specificity. In summary, MPTE-modified oligonucleotides are a promising DNA analog applied to PCR primers and probes to enhance the specificity and to provide more precise detection results for various applications, such as for genetic diagnosis. In summary, two common DNA polymerases we tested could successfully recognize the MPTE-modified primers and probes. Under the optimal operating conditions, MPTE modification has the ability to improve the discrimination of single nucleotide polymorphism by increasing the ΔTm of the perfect match and mismatch sequences and to provide more precise detection results for various applications, such as genetic diagnosis.

Isothermal titration calorimetry for characterization of recombinant proteins.

Isothermal titration calorimetry is widely used to measure the affinities and enthalpies of interaction between proteins and/or small molecules. The quantitative nature of the technique is especially useful in the characterization of recombinant proteins while determining the fraction of protein capable of binding a specific ligand and thus the protein purity. The revealed thermodynamic information sheds light on the binding mechanism, important for the targeted drug design of the biologics. Here we show examples how, together with the thermal shift assay, combination of both techniques enables characterization of protein stability and ligand binding. Furthermore, the binding-linked reactions that strongly affect the observed thermodynamic parameters and must be dissected to obtain the intrinsic parameters that are necessary for the structure-based rational drug design are being demonstrated using inhibitors of Hsp90, an anticancer target protein.