Recent Progress on Optical Biosensors Developed for Nucleic Acid Detection Related to Infectious Viral Diseases.

Optical biosensors have many advantages over traditional analytical methods. They enable the identification of several biological and chemical compounds directly, instantly, and without the need of labels. Their benefits include excellent specificity, sensitivity, compact size, and low cost. In this review, the main focus is placed on the nucleic acid-based optical biosensor technologies, including colorimetric, fluorescence, surface plasmon resonance (SPR), Evanescent-Wave Optical, Fiber optic and bioluminescent optical fibre. The fundamentals of each type of biosensor are briefly explained, and particular emphasis has been placed on the achievements which have been gained in the last decade on the field of diagnosis of infectious viral diseases. Concluding remarks concerning the perspectives of further developments are discussed.

Zip Nucleic Acid-Based Genomagnetic Assay for Electrochemical Detection of microRNA-34a.

Zip nucleic acid (ZNA)-based genomagnetic assay was developed herein for the electrochemical detection of microRNA-34a (miR-34a), which is related to neurological disorders and cancer. The hybridization between the ZNA probe and miR-34a target was performed in the solution phase; then, the resultant hybrids were immobilized onto the surface of magnetic beads (MBs). After magnetic separation, the hybrids were separated from the surface of MBs and then immobilized on the surface of pencil graphite electrodes (PGEs). In the case of a full-match hybridization, the guanine oxidation signal was measured via the differential pulse voltammetry (DPV) technique. All the experimental parameters that influenced the hybridization efficiency (i.e., hybridization strategy, probe concentration, hybridization temperature, etc.) were optimized. The cross-selectivity of the genomagnetic assay was tested against two different miRNAs, miR-155 and miR-181b, individually as well as in mixture samples. To show the applicability of the ZNA-based genomagnetic assay for miR-34a detection in real samples, a batch of experiments was carried out in this study by using the total RNA samples isolated from the human hepatocellular carcinoma cell line (HUH-7).

Aptasensor for Impedimetric Detection of Lysozyme.

The impedimetric detection of a protein, lysozyme (LYS), was carried out herein by aptamer-immobilized single-use pencil graphite electrodes (PGEs). The aptamer was immobilized onto electrochemically activated surface of electrode without using any chemical agents, or any types of nanomaterials. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were applied to analyze the electrochemical behavior of unmodified PGE and aptamer immobilized PGE. The interaction of aptamer with its target protein, LYS, was then investigated by EIS. The limit of detection for LYS was found to be 1.44 µg/mL (equals to 100.65 nM). The developed aptasensor specific to LYS presented high selectivity against to bovine serum albumin and thrombin.

Low-Cost High-Resolution Potentiostat for Electrochemical Detection of Nucleic Acids and Biomolecular Interactions.

A handheld USB-powered instrument developed for the electrochemical detection of nucleic acids and biomolecular interactions is presented. The proposed instrument is capable of scanning ± 2.25 V while measuring currents up to ±10 mA, with a minimum current resolution of 6.87 pA. Therefore, it is suitable for nucleic acid sensors, which have high background currents. A low-cost microcontroller with an on-chip 16-bit analog-to-digital converter, 12-bit digital-to-analog converter, and a built-in USB controller were used to miniaturize the system. The offset voltages and gain errors of the analog peripherals were calibrated to obtain a superior performance. Thus, a similar performance to those of the market-leader potentiostats was achieved, but at a fraction of their cost and size. The performance of the application of this proposed architecture was tested successfully and was found to be similar to a leading commercial device through a clinical application in the aspects of the detection of nucleic acids, such as calf thymus ssDNA and dsDNA, and their interactions with a protein (BSA) by using single-use graphite electrodes in combination with the differential pulse voltammetry technique.

An electrochemical assay for sensitive detection of Acinetobacter baumannii gene.

A new genosensor, which allows sensitive and selective detection of Acinetobacter baumannii gene sequence was developed herein. In this assay, capture probe of Acinetobacter baumannii was immobilized on the surface of chitosan modified single-use pencil graphite electrodes (c-PGEs) to obtain Acinetobacter baumannii genosensor. Then, Acinetobacter baumannii target DNA sequence was recognized after solid-state hybridization on c-PGE genosensor by measuring guanine signal via differential pulse voltammetry (DPV). In order to improve hybridization efficiency, experimental parameters affecting all assay steps are studied and the analytical performance of the genosensor was tested. The low limit of detection (LOD) for Acinetobacter baumannii target DNA sequence was obtained as 1.86 nM with developed genosensor. The selectivity of the proposed assay was then tested in the presence of 1-base mismatch, or two different type of non-complementary sequences and no interference effect was observed. The proposed electrochemical assay protocol is easy, convenient, and rapid which can be a decent alternative to existing methods.

Graphene-Oxide and Ionic Liquid Modified Electrodes for Electrochemical Sensing of Breast Cancer 1 Gene.

Graphene-oxide and ionic liquid composite-modified pencil graphite electrodes (GO-IL-PGEs) were developed and used as a sensing platform for breast cancer 1 (BRCA1) gene detection. The characterization of GO-IL modified electrodes was executed by scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The nucleic-acid hybridization was monitored by a differential pulse voltammetry (DPV) technique by directly measuring the guanine oxidation signal without using any indicator. The effects of the IL concentration, the probe concentration, and the hybridization time were optimized to the biosensor response. The limit of detection (LOD) was calculated in the concentration range of 2-10 µg/mL for the BRCA1 gene and found to be 1.48 µg/mL. The sensitivity of the sensor was calculated as 1.49 µA mL/µg cm2. The developed biosensor can effectively discriminate the complementary target sequence in comparison to a three-base-mismatched sequence or the non-complementary one.

Impedimetric detection of miRNA biomarkers using paper-based electrodes modified with bulk crystals or nanosheets of molybdenum disulfide.

Paper-based electrodes modified with molybdenum disulfide (MoS2) in the form of bulk crystals or exfoliated nanosheets were developed and used as a biosensing platform for the impedimetric detection of miRNAs (miRNA-155 and miRNA-21) related to early diagnosis of lung cancer. For this purpose, MoS2 crystals or nanosheets were used for the modification of the working electrode area of paper-based platform for the first time in this study. The proposed assay offers sensitive and selective detection of microRNAs by electrochemical impedance spectroscopy (EIS) technique. The entire assay, both the electrode modification and the miRNA detection being completed in 30 min and a single sample droplet (5 µL) was enough to cover working electrode area which enabled analysis in low sample volumes. The limits of detection (LOD) for miRNA-21 and miRNA-155 were calculated both in buffer and fetal bovine serum media. It is found that the LOD is varying between 1 and 200 ng/mL. In comparison to nanosheets, a larger electroactive surface area was obtained with bulk MoS2 resulting in lower LOD values on miRNA detection. The paper-based electrodes showed high specificity towards their target sequences. Moreover, they effectively discriminated single base mismatched non-target sequences. The advantages of these MoS2 paper based electrodes include high sensitivity, and low-cost provide great potential for improved monitoring of miRNA biomarkers even in artificial serum media.

Detection of Senecionine in Dietary Sources by Single-Use Electrochemical Sensor.

Pyrrolizidine alkaloids (PAs) are produced by plants as secondary compounds that are the most widely distributed natural toxins. There have been many cases of human toxicity caused by consumption of toxic plant species, as herbal teas and grain or grain products contaminated with PA-containing seeds have been reported. Companies that produce dried spices and tea leaves should examine the PA level in their products. For the first time in the literature, a simple and inexpensive electrochemical assay based on a single-use sensor was introduced for quantitative determination of senecionine (SEN) in the most frequently contaminated food sources. SEN was immobilized on a pencil graphite electrode surface by the passive adsorption technique. Differential pulse voltammetry (DPV) was used to evaluate the oxidation signal of SEN, which was observed to be around +0.95 V. The oxidation signal was specific to the SEN in the sample, and the current value was proportional to its concentration. The selectivity of our assay was also tested in the presence of other similar PAs such as intermedine, lycopsamine, and heliotrine. The detection limit is calculated by developed assay and found to be 5.45 µg/mL, which is an acceptable concentration value of SEN occurring at toxic levels for consumers. As an application of the developed sensor in food products, the electrochemical detection of SEN was successfully performed in flour and herbal tea products.

Paper-Based Electrochemical Biosensors for Voltammetric Detection of miRNA Biomarkers Using Reduced Graphene Oxide or MoS2 Nanosheets Decorated with Gold Nanoparticle Electrodes.

Paper-based biosensors are considered simple and cost-efficient sensing platforms for analytical tests and diagnostics. Here, a paper-based electrochemical biosensor was developed for the rapid and sensitive detection of microRNAs (miRNA-155 and miRNA-21) related to early diagnosis of lung cancer. Hydrophobic barriers to creating electrode areas were manufactured by wax printing, whereas a three-electrode system was fabricated by a simple stencil approach. A carbon-based working electrode was modified using either reduced graphene oxide or molybdenum disulfide nanosheets modified with gold nanoparticle (AuNPs/RGO, AuNPs/MoS2) hybrid structures. The resulting paper-based biosensors offered sensitive detection of miRNA-155 and miRNA-21 by differential pulse voltammetry (DPV) in only 5.0 µL sample. The duration in our assay from the point of electrode modification to the final detection of miRNA was completed within only 35 min. The detection limits for miRNA-21 and miRNA-155 were found to be 12.0 and 25.7 nM for AuNPs/RGO and 51.6 and 59.6 nM for AuNPs/MoS2 sensors in the case of perfectly matched probe-target hybrids. These biosensors were found to be selective enough to distinguish the target miRNA in the presence of single-base mismatch miRNA or noncomplementary miRNA sequences.

Electrochemical Investigation of Curcumin-DNA Interaction by Using Hydroxyapatite Nanoparticles-Ionic Liquids Based Composite Electrodes.

Hydroxyapatite nanoparticles (HaP) and ionic liquid (IL) modified pencil graphite electrodes (PGEs) are newly developed in this assay. Electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and cyclic voltammetry (CV) were applied to examine the microscopic and electrochemical characterization of HaP and IL-modified biosensors. The interaction of curcumin with nucleic acids and polymerase chain reaction (PCR) samples was investigated by measuring the changes at the oxidation signals of both curcumin and guanine by differential pulse voltammetry (DPV) technique. The optimization of curcumin concentration, DNA concentration, and the interaction time was performed. The interaction of curcumin with PCR samples was also investigated by gel electrophoresis.

Paper-based electrode assemble for impedimetric detection of miRNA.

In the present work, a paper-based electrode assemble was developed and implemented to detect target microRNA 155 (miRNA 155) via electrochemical impedance spectroscopy (EIS) measurements. In this concept, gold nanoparticles (AuNPs) modified paper based electrode assemble system (AuNP-PE) was designed, and characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and EIS measurements. The impedimetric detection of miRNA 155 was performed by measuring the fractional change at the charge transfer resistance (Rct). The detection limits were found as 33.8 nM in PBS and 93.4 nM in fetal bovine serum (FBS) medium, respectively. The selectivity of the proposed assay was tested against to non-complementary (NC) and mismatch (MM) miRNA sequences in the presence of mixture sample containing miRNA:NC (1:1) and miRNA:MM (1:1) in PBS (pH 7.40) or FBS. The analytical performance and the selectivity of impedimetric biosensor were also tested in FBS.

Impedimetric Sensing of Factor V Leiden Mutation by Zip Nucleic Acid Probe and Electrochemical Array.

A carbon nanofiber enriched 8-channel screen-printed electrochemical array was used for the impedimetric detection of SNP related to Factor V Leiden (FV Leiden) mutation, which is the most common inherited form of thrombophilia. FV Leiden mutation sensing was carried out in three steps: solution-phase nucleic acid hybridization between zip nucleic acid probe (Z-probe) and mutant type DNA target, followed by the immobilization of the hybrid on the working electrode area of array, and measurement by electrochemical impedance spectroscopy (EIS). TArzhe selectivity of the assay was tested against mutation-free DNA sequences and synthetic polymerase chain reaction (PCR) samples. The developed biosensor was a trustful assay for FV Leiden mutation diagnosis, which can effectively discriminate wild type and mutant type even in PCR samples.

Voltammetric detection of miRNA hybridization based on electroactive indicator-cobalt phenanthroline.

The indicator-based nucleic acid detection protocol is one of the major approaches to monitor the sequence-selective nucleic acid hybridization-mediated recognition events in biochemical analysis. The metal complex, cobalt phenanthroline, [Co(phen)33+], which is one of the electroactive indicators, interacts more with double stranded nucleic acids via intercalation. Thus, this interaction permits an increase at the electrochemical signal of [Co(phen)33+]. In our study, the interaction of metal complex, [Co(phen)33+] with nucleic acids was examined using pencil graphite electrodes (PGEs) in combination with differential pulse voltammetry (DPV) technique. The voltammetric detection of miRNA-34a was investigated based on the changes at the electrochemical signal of [Co(phen)33+] under optimized experimental conditions; such as accumulation potentialof metal complex and DNA probe concentration, hybridization time, target miRNA concentration. Furthermore, the selectivity of electrochemical miRNA-34a biosensor was studied in contrast to different miRNAs. The applicability of indicator-based biosensor specific to miRNA-34a was also presented by using total RNA samples.

Electrochemical Detection of Solution Phase Hybridization Related to Single Nucleotide Mutation by Carbon Nanofibers Enriched Electrodes.

In the present study, a sensitive and selective impedimetric detection of solution-phase nucleic acid hybridization related to Factor V Leiden (FV Leiden) mutation was performed by carbon nanofibers (CNF) modified screen printed electrodes (SPE). The microscopic and electrochemical characterization of CNF-SPEs was explored in comparison to the unmodified electrodes. Since the FV Leiden mutation is a widespread inherited risk factor predisposing to venous thromboembolism, this study herein aimed to perform the impedimetric detection of FV Leiden mutation by a zip nucleic acid (ZNA) probe-based assay in combination with CNF-SPEs. The selectivity of the assay was then examined against the mutation-free DNA sequences as well as the synthetic PCR samples.

ZNA probe immobilized single-use electrodes for impedimetric detection of nucleic acid hybridization related to single nucleotide mutation.

The development of a low-cost and disposable biosensing technologies has received a great interest of healthcare for the sensitive and reliable detection of single nucleotide mutation related to single nucleotide polymorphisms (SNPs). In the present study, an impedimetric biosensing platform based on zip nucleic acids (ZNA) was developed for the sensitive detection of Factor V Leiden (FV Leiden) mutation. After optimization of experimental parameters, the sequence selective hybridization between ZNA probe and target related to FV Leiden mutation was evaluated via electrochemical impedance spectroscopy technique (EIS) by measuring changes at the charge transfer resistance, Rct. Sensitive and selective impedimetric analysis was performed using carbon nanofiber (CNF) modified screen printed electrodes (SPE) and multi-channel screen printed array of electrodes (MULTIx8 CNF-SPE) resulting in a relatively shorter time in comparison to conventional methods. The selectivity of ZNA probe to mutation-free DNA sequences was also investigated. The applicability of single-use ZNA biosensor was also tested in synthetic PCR samples containing a single base mutation.

Enzymatic/Immunoassay Dual-Biomarker Sensing Chip: Towards Decentralized Insulin/Glucose Detection.

Performing bioassay formats based on enzyme and antibody recognition reactions with a single detection chip remains an unmet challenge owing to the different requirements of such bioassays. Herein, we describe a dual-marker biosensor chip, integrating enzyme and antibody-based assays for simultaneous electrochemical measurements of insulin (I) and glucose (G). Simultaneous G/I sensing has been realized by addressing key fabrication and operational challenges associated with the different assay requirements and surface chemistry. The I immunosensor relies on a peroxidase-labeled sandwich immunoassay, while G is monitored through reaction with glucose oxidase. The dual diabetes biomarker chip offers selective and reproducible detection of picomolar I and millimolar G concentrations in a single microliter sample droplet within less than 30 min, including direct measurements in whole blood and saliva samples. The resulting integrated enzymatic-immunoassay biosensor chip opens a new realm in point-of-care multiplexed biomarker detection.

Investigation of Vipera Anatolica Venom Disintegrin via Intracellular Uptake with Radiolabeling Study and Cell-Based Electrochemical Biosensing Assay.

Snake venoms are a natural biological source that has potential therapeutic value with various protein compounds. Disintegrins originally were discovered as a family of proteins from snake venoms composed of cysteine rich low molecular weight polypeptides. Disintegrins exhibit specific binding and higher affinity toward integrin with potential inhibition of function. Trans-membrane receptors of the integrin family may involve in many pathological conditions such as inflammation and tumor progression with important processes related to invasion and migration. Since disintegrins have the ability to bind to integrins, they could be used for cancer detection and treatment, and in monitoring of therapy in select cancer types. The main purpose of the study is to investigate disintegrin containing Vipera anatolica (VAT) crude venom potential for radiolabeling and intracellular uptake as well as electrochemical biosensing assay against U87MG human brain glioblastoma cells. For this purpose, VAT crude venom containing U87MG cell-specific disintegrin was investigated in terms of radiolabeling and intracellular uptake as well as electrochemical biosensing assay in comparison with echistatin (ECT) disintegrin in cells. The interaction between VAT crude venom and ECT with HEK293 human non-tumorigenic embryonic kidney cells and glioblastoma U87MG cells was electrochemically investigated using pencil graphite electrodes (PGEs). The interaction of the VAT crude venom and ECT with HEK293 and U87MG cells was detected according to the changes in oxidation signals. Then, VAT crude venom and echistatin were labeled with 131I via iodogen method. Intracellular uptakes of radiolabeled molecules were investigated in U87MG cell line. 131I-VAT can be an agent for imaging of glioblastoma cancer. Further work will focus on the production of large quantities of pure VAT disintegrin with a biotechnological approach to improving imaging agent.

Magnetic beads assay based on Zip nucleic acid for electrochemical detection of Factor V Leiden mutation.

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation among people. Development of reliable methods for the detection of SNP is crucial in aspects of molecular diagnosis and personalized medicine. In our study, a genomagnetic assay in combination with zip nucleic acid (ZNA) for electrochemical detection of SNP related to Factor V Leiden mutation. For the first time in the literature, a new generation nucleic acid; ZNA was applied herein for electrochemical monitoring of nucleic acid hybridization. Streptavidin coated magnetic beads (MBs) were used for preparation of samples containing ZNA-DNA hybrid and accordingly, the guanine signal was measured as a response of hybridization related to Factor V Leiden mutation by carbon nanofibers (CNF) modified screen printed electrodes (SPE) and multi-channel screen printed array of electrodes (CNF-MULTI SPEx8). The detection limit (DL) was found to be 3.79 µg/mL (376 nM) and, 11.63 µg/mL (1.624 µM), respectively by CNF-SPE and CNF-MULTI SPEx8. The selectivity of ZNA probe to mutation-free DNA sequences was also investigated in contrast to DNA probe. The applicability of ZNA based magnetic beads assay to sequence selective hybridization related to Factor V Leiden was also tested in synthetic PCR samples.

An Impedimetric Biosensor Based on Ionic Liquid-Modified Graphite Electrodes Developed for microRNA-34a Detection.

In the present work, an impedimetric nucleic acid biosensor has been designed for the purpose of detection of microRNA (miRNA). Ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL))-modified chemically activated pencil graphite electrodes (PGEs) were used for the sensitive and selective detection of miRNA-34a. After covalent activation of the PGE surface using covalent agents (CAs), the ionic liquid (IL) was immobilized onto the surface of the chemically activated PGE by passive adsorption. The electrochemical and microscopic characterization of the IL/CA/PGEs was performed by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and scanning electron microscopy (SEM). DNA probe concentration, miRNA target concentration, and also the hybridization time and wet adsorption time were optimized by using the EIS technique. Then, the hybridization occurred between specific DNA probes and miRNA-34a was immobilized onto the surface of the IL/CA/PGEs. The impedimetric detection of miRNA-DNA hybrid was performed by EIS. The detection limit (DL) was calculated in a linear concentration range of 2⁻10 µg/mL miRNA-34a target, and it was found to be 0.772 µg/mL (109 nM) in phosphate buffer solution (PBS) and 0.826 µg/mL (117 nM) in diluted fetal bovine serum (FBS). The selectivity of impedimetric biosensor for miRNA-34a was also tested against to other non-complementary miRNA sequences both in buffer media, or diluted FBS.

Electrochemical detection of interaction between capsaicin and nucleic acids in comparison to agarose gel electrophoresis.

In this study, the biomolecular interaction occurred between nucleic acids and Capsaicin (CPS), the active compound in chilli peppers, which has been reported to have anti-carcinogenic properties, was investigated for the first time herein using disposable electrochemical biosensor. It is aimed to perform the surface-confined interaction between CPS and different types of nucleic acids and under this aim, the experimental conditions were optimized; such as, the concentration of CPS and DNA, DNA immobilization time and interaction time etc. The detection limit of DNA was estimated based on guanine oxidation signal in the linear concentration range of DNA from 1 to 5 µg/mL, and it was found to be 0.62 µg/mL. The effect of time-dependent manner from 1 min to 30 min on the interaction of CPS with nucleic acids was explored upon to the changes at guanine signal coming from double stranded DNA and cDNA as well as PCR samples. The interaction of CPS with double stranded DNA was also determined by agarose gel electrophoresis.