DNA Immobilization Research Articles

Programmable graphene-based microfluidic sensor for DNA detection

This study presents the development of a lab-on-a-chip (LoC) by integrating a graphene field-effect transistor (FET) chip with a programmable microfluidic device for DNA detection. The real-time biochemical events on the graphene FET chip were monitored through Dirac voltage shift data from the portable graphene curve reader with changes dependent on the fluidic flow into the sensing interface by a fully automated programmable microfluidic system. High sensitivity with high reliability can be obtained with a nine-graphene sensor layout on a single chip. The portable graphene curve reader also provides a tunable electrical parameter setup and straightforward data acquisition. Fluidic control was performed through a multi-position valve, allowing sequential commands for liquid injection into the polydimethylsiloxane (PDMS) flow cell mounted on the sensing chip. The flow cell design with impinging jet geometry and the microfluidic system packaging offer high precision and portability as a less laborious and low-cost sensing setup. The merged system allows for various functionalities, including probe DNA (pDNA) immobilization, a blocking step, and DNA hybridization with stable signal output autonomously, even in a long-run experimental setup. As a DNA sensor, the proposed prototype has demonstrated a high sensitivity of ~44 mV/decade of target DNA concentration, with an outstanding limit of detection (LoD) of ~0.642 aM, making it one of the most sensitive sensors reported up to date. The programmable device has demonstrated essential versatilities for biomolecular detection in a fully portable and automated platform.

Chiral Sum Frequency Generation Spectroscopy Detects Double-Helix DNA at Interfaces.

Many DNA-based technologies involve the immobilization of DNA and therefore require a fundamental understanding of the DNA structure-function relationship at interfaces. We present three immobilization methods compatible with chiral sum frequency generation (SFG) spectroscopy at interfaces. They are the "anchor" method for covalently attaching DNA on a glass surface, the "island" method for dropcasting DNA on solid substrates, and the "buoy" method using a hydrocarbon moiety for localizing DNA at the air-water interface. Although SFG was previously used to probe DNA, the chiral and achiral SFG responses of single-stranded and double-stranded DNA have not been compared systemically. Using the three immobilization methods, we obtain the achiral and chiral C-H stretching spectra. The results introduce four potential applications of chiral SFG. First, chiral SFG gives null response from single-stranded DNA but prominent signals from double-stranded DNA, providing a simple binary readout for label-free detection of DNA hybridization. Second, with heterodyne detection, chiral SFG gives an opposite-signed spectral response useful for distinguishing native (D-) right-handed double helix from non-native (L-) left-handed double helix. Third, chiral SFG captures the aromatic C-H stretching modes of nucleobases that emerge upon hybridization, revealing the power of chiral SFG to probe highly localized molecular structures within DNA. Finally, chiral SFG is sensitive to macroscopic chirality but not local chiral centers and thus can detect not only canonical antiparallel double helix but also other DNA secondary structures, such as a poly-adenine parallel double helix. Our work benchmarks the SFG responses of DNA immobilized by the three distinct methods, building a basis for new chiral SFG applications to solve fundamental and biotechnological problems.

Test Strip Platform Spin-Off for Telomerase Activity Detection: Development of an Electrochemical Biosensor.

Telomerase overexpression has been associated directly with cancer, and the enzyme itself is recognized within the scientific community as a cancer biomarker. BIDEA’s biosensing strip (BBS) is an innovative technology capable of detecting the presence of telomerase activity (TA) using electrochemical impedance spectroscopy (EIS). This BBS is an interdigital gold (GID) electrode array similar in size and handling to a portable glucose sensor. For the detection of the biomarker, BBS was modified by the immobilization of a telomere-like single strand DNA (ssDNA) on its surface. The sensor was exposed to telomerase-positive extract from commercially available cancer cells, and the EIS spectra were measured. Telomerase recognizes the sequence of this immobilized ssDNA probe on the BBS, and the reverse transcription process that occurs in cancer cells is replicated, resulting in the ssDNA probe elongation. This surface process caused by the presence of TA generates changes in the capacitive process on the electrode array microchip surface, which is followed by EIS as the sensing tool and correlated with the presence of cancer cells. The telomerases’ total cell extraction protocol results demonstrate significant changes in the charge-transfer resistance (Rct) change rate after exposure to telomerase-positive extract with a detection limit of 2.94 × 104 cells/mL. Finally, a preliminary study with a small set of “blind” uterine biopsy samples suggests the feasibility of using the changes in the Rct magnitude change rate (Δ(ΔRct/Rcti)/Δt) to distinguish positive from negative endometrial adenocarcinoma samples by the presence or absence of TA.

Regenerable bead-based microfluidic device with integrated thin-film photodiodes for real-time monitoring of DNA detection

Recently, systems for rapid diagnosis of diseases and infections have motivated the development of new DNA detection platforms. However, these platforms have failed to reach the market mainly because of the potential risk of misdiagnosis. Thus, developing a system with an effective internal control, guaranteeing the veracity of the result, could help overcome this barrier. The possibility to regenerate the microfluidic biosensing system would allow the implementation of the aforementioned internal control and the system's reusability, resulting in cost reduction. Nanoporous microbead-based microfluidic systems coupled with on-chip signal transduction can potentially allow significant enhancement of sensitivity for biosensing applications. In this paper, we report a regenerable bead-based microfluidic device that employs integrated thin-film photodiodes for real-time monitoring of DNA molecular recognition. The molecular identification of target DNA and complementary DNA is accomplished, followed by regeneration of the chip using 0.3 M of NaOH and control with a non-complementary target DNA. The best condition for immobilization of probe DNA to allow regeneration of the device was studied, and limits of detection of 1.2 nM and 3.1 nM were obtained for target DNA in buffer and human plasma, respectively, detected by fluorescence assays. Moreover, a chemiluminescence strategy provided the highest sensitivity to detect target DNA with a limit of detection of 78.5 pM. High-sensitivity assay cycles could be performed without significant loss of probe DNA, demonstrating the potential for the system's reusability, portability, and reproducibility.

Temperature–regulated non-monotonic behavior of DNA immobilization on poly(N–isopropylacrylamide) (PNIPAm)–grafted surface

Immobilizing single–stranded DNA (ssDNA) probes onto transducer surface is the initial step to construct DNA biosensors, and its surface density is crucial for the performance of the designed sensors. Here, we report the surface density of Cy3–labeled DNA probes immobilized on poly(N–isopropylacrylamide) (PNIPAm)–grafted indium–tin oxide (ITO) surface shows temperature–regulated behavior by combining experiment and molecular theory. DNA immobilization gets hindered by the hydrophilic PNIPAm layer grafted on ITO surface at 25 oC, and the surface density of DNA probes gets monotonically decreased as PNIPAm surface coverage rises. However, DNA immobilization shows non–monotonic variation depending on the PNIPAm surface coverage at 45 oC. More specifically, the immobilized DNA probes get facilitated by the hydrophobic PNIPAm layer at low surface coverage, and the largest surface density of DNA probes reaches 1.4–fold that of the bare ITO surface, but DNA immobilization gets blocked by the hydrophobic PNIPAm layer at high surface coverage. The temperature–regulated behavior of DNA immobilization on PNIPAm–grafted surface may be exploited for design of thermo–responsive DNA biosensors and self–assemblies based on DNA probes.

Photo-Assisted Robust Anti-Interference Self-Powered Biosensing of MicroRNA Based on Pt-S Bonds and the Inorganic-Organic Hybridization Strategy.

Photo-assisted biofuel cell-based self-powered biosensors (PBFC-SPBs) possess the advantages of no need for external power supply, ease of sensing design, and simple instruments. In this work, a robust anti-interference PBFC-SPB for microRNA detection was constructed based on the Pt-S bond and the inorganic-organic hybridization strategy. The organic semiconductor [6,6]-phenyl-C61-butyric acid methylester@anthraquinone (PCBM@anthraquinone) served as an efficient light-harvesting material, and gold nanoparticle@Pt (AuNP@Pt) nanomaterials were immobilized on the surface via electrostatic adsorption for the binding of DNA. Notably, compared to Au-S bonds for DNA immobilization, the Pt-S bond exhibited better anti-interference ability. Ingeniously, cadmium sulfide quantum dots (CdS QDs) were close to the PCBM@anthraquinone substrate electrode to form sensitization structures, which was beneficial to enhance the photocurrent signal. Combining with the laccase-mimicking activity Cu2+/carbon nanotubes (Cu2+/CNTs) cathode, the PBFC-SPB for microRNA detection was achieved. Once the target existed, the identical sequence complementary microRNA would make DNA2/CdS dissociate and break away from the electrode, leading to a low signal. The linear detection range was 10 fM-100 pM, with the limit of determination of 2.4 fM (3S/N). The as-proposed strategy not only paves a new way for the design of photoelectrochemical biosensing but also opens a door for the construction of robust anti-interference bioassay for microRNA detection.

A new electrochemical DNA biosensor for determination of anti-cancer drug chlorambucil based on a polypyrrole/flower-like platinum/NiCo2O4/pencil graphite electrode.

In the current study, DNA immobilization was performed on pencil graphite (PG) modified with a polypyrrole (PPy) and flower-like Pt/NiCo2O4 (FL-Pt/NiCo2O4) nanocomposite, as a new sensitive electrode to detect chlorambucil (CHB). Energy dispersive X-ray (EDX) analysis, X-ray diffraction (XRD) and scanning electron microscopy (SEM) techniques were employed to characterize the synthesized FL-Pt/NiCo2O4 and PPy/FL-Pt/NiCo2O4 nanocomposites. Moreover, differential pulse voltammetry (DPV) was selected to assess the guanine and adenine electrochemical responses on the DNA sensor. The CHB determination was performed using the maximum currents towards adenine and guanine in the acetate buffer solution (ABS). According to the results, ds-DNA/PPy/FL-Pt/NiCo2O4/PGE was able to detect the different concentrations of CHB in the range between 0.018 and 200 μM, with a detection limit of (LOD) of 4.0 nM. The new biosensor was also exploited for CHB determination in real samples (serum, urine and drug), the results of which revealed excellent recoveries (97.5% to 103.8%). Furthermore, the interaction between ds-DNA and CHB was studied using electrochemistry, spectrophotometry and docking whose outputs confirmed their effective interaction.

Cell-Free Gene Expression from DNA Brushes.

Linear double-stranded DNA polymers coding for synthetic genes immobilized on a surface form a brush as a center for cell-free gene expression, with DNA density 102-103 fold higher than in bulk solution reactions. A brush localizes the transcription-translation machinery in cell extracts or in cell-free reconstituted reactions from purified components, creating a concentrated source of RNA and proteins. Newly synthesized molecules can form circuits regulating gene expression in the same brush or adjacent ones. They can also assemble into functional complexes and machines such as ribosomal units, then analyzed by capture on prepatterned antibodies or by cascaded reactions. DNA brushes are arranged as a single center or multiple ones on a glass coverslip, in miniaturized compartments carved in silicon wafers, or in elastomeric microfluidic devices. Brushes create genetically programmable artificial cells with steady-state dynamics of protein synthesis. Here, we provide the basic procedure for surface patterning, DNA immobilization, capture of protein products on antibody traps and fluorescent imaging. The method of DNA brush surface patterning enables simple parallelization of cell-free gene expression reactions for high throughput studies with increased imaging sensitivity.

Fluorescence Detection of miRNA-21 Using Au/Pt Bimetallic Tubular Micromotors Driven by Chemical and Surface Acoustic Wave Forces.

In this study, surface acoustic wave (SAW) systems are described for the removal of molecules that are unbound to micromotors, thereby lowering the detection limit of the cancer-related biomarker miRNA-21. For this purpose, in the first step, mass production of the Au/Pt bimetallic tubular micromotor was performed with a simple membrane template electrodeposition. The motions of catalytic Au/Pt micromotors in peroxide fuel media were analyzed under the SAW field effect. The changes in the micromotor speed were investigated depending on the type and concentration of surfactants in the presence and absence of SAW streaming. Our detection strategy was based on immobilization of probe dye-labeled single-stranded probe DNA (6-carboxyfluorescein dye-labeled-single-stranded DNA) to Au/Pt micromotors that recognize target miRNA-21. Before/after hybridization of miRNA-21 (for both w/o SAW and SAW streaming conditions), the changes in the speed of micromotors and their fluorescence intensities were studied. The response of fluorescence intensities was observed to be linearly varied with the increase of the miRNA-21 concentration from 0.5 to 5 nM under both w/o SAW and with SAW. The resulting fluorescence sensor showed a limit of detection of 0.19 nM, more than 2 folds lower compared to w/o SAW conditions. Thus, the sensor and behaviors of Au/Pt tubular micromotors were improved by acoustic removal systems.

Equipment-free, salt-mediated immobilization of nucleic acids for nucleic acid lateral flow assays

As the world has been facing several deadly virus crises, including Zika virus disease, Ebola virus disease, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19), lateral flow assays (LFAs), which require minimal equipment for point-of-care of viral infectious diseases, are garnering much attention. Accordingly, there is an increasing demand to reduce the time and cost required for manufacturing LFAs. The current study introduces an equipment-free method of salt-mediated immobilization of nucleic acids (SAIoNs) for LFAs. Compared to general DNA immobilization methods such as streptavidin–biotin, UV-irradiation, and heat treatment, our method does not require special equipment (e.g., centrifuge, UV-crosslinker, heating device); therefore, it can be applied in a resource-limited environment with reduced production costs. The immobilization process was streamlined and completed within 30 min. Our method improved the color intensity signal approximately 14 times compared to the method without using SAIoNs and exhibited reproducibility with the long-term storage stability. The proposed method can be used to detect practical targets (e.g., SARS-CoV-2) and facilitates highly sensitive and selective detection of target nucleic acids with multiplexing capability and without any cross-reactivity. This novel immobilization strategy provides a basis for easily and inexpensively developing nucleic acid LFAs combined with various types of nucleic acid amplification.

A DNA Nanoflower-Assisted Separation-Free Nucleic Acid Detection Platform with a Commercial Pregnancy Test Strip.

There is a constant drive for affordable point-of-care testing (POCT) technologies for the detection of infectious human diseases. Herein, we report a simple platform for DNA detection that takes advantage of four techniques: commercially available pregnancy test strips (PTS), amplicon generation via loop-mediated isothermal amplification (LAMP), toehold-mediated strand displacement, and noncovalent immobilization of DNA on paper surface with DNA nanoflowers. This simple, separation-free platform is highly specific, as demonstrated with the detection of rtL180M, a single-nucleotide polymorphism observed in hepatitis B virus (HBV) associated with antiviral drug resistance. It is very sensitive, capable of detecting the targeted mutation at 2 copies μL-1 . It is able to correctly identify the unmutated and rtL180M genome types of HBV in clinical samples. Given its wide adaptability, we expect this platform can be easily modified for the detection of genetic variations associated with various pathogens and human diseases.

A DNA Nanoflower‐Assisted Separation‐Free Nucleic Acid Detection Platform with a Commercial Pregnancy Test Strip

AbstractThere is a constant drive for affordable point‐of‐care testing (POCT) technologies for the detection of infectious human diseases. Herein, we report a simple platform for DNA detection that takes advantage of four techniques: commercially available pregnancy test strips (PTS), amplicon generation via loop‐mediated isothermal amplification (LAMP), toehold‐mediated strand displacement, and noncovalent immobilization of DNA on paper surface with DNA nanoflowers. This simple, separation‐free platform is highly specific, as demonstrated with the detection of rtL180M, a single‐nucleotide polymorphism observed in hepatitis B virus (HBV) associated with antiviral drug resistance. It is very sensitive, capable of detecting the targeted mutation at 2 copies μL−1. It is able to correctly identify the unmutated and rtL180M genome types of HBV in clinical samples. Given its wide adaptability, we expect this platform can be easily modified for the detection of genetic variations associated with various pathogens and human diseases.

Metal-Mediated Polydopamine Nanoparticles-DNA Nanomachine Coupling Electrochemical Conversion of Metal-Organic Frameworks for Ultrasensitive MicroRNA Sensing.

The development of a robust sensing platform with an efficient probe assembly, and ingenious signal conversion is of great significance for bioanalytical application. In this work, a multipedal polydopamine nanoparticles-DNA (PDANs-DNA) nanomachine coupling electrochemical-driven metal-organic frameworks (MOFs) conversion-enabled biosensing platform was constructed. The PDANs-DNA nanomachine was designed based on Ca2+-mediated DNA adsorption and target-triggered catalytic hairpin assembly on PDANs, which not only maintained the DNA immobilization simplicity but also possessed a high walking efficiency. PDANs-DNA nanomachine could walk fast on the electrode via multiple legs under exonuclease III driving, resulting in the formation of DNA dendrimers through two hairpins assembly. The MOFs (Fe-MIL-88-NH2) probe was decorated on the DNA dendrimers to act as a porous metal precursor and converted into electroactive Prussian Blue by a controlled electrochemical approach, which was a facile, simple, and room-temperature approach compared with the commonly employed MOFs conversion methods. Using microRNA-21 (miRNA-21) as the model target, the proposed biosensor achieved miRNA-21 detection ranging from 10 aM to 10 pM with the detection limit of 5.8 aM. The proposed strategy presented a highly efficient walking platform with the ingenious electrochemical conversion of MOFs, providing more options for the design of an electrochemical platform and holding potential applications in clinical analysis and disease diagnosis.

Preparation of E. coli RNA polymerase transcription elongation complexes by selective photoelution from magnetic beads

In vitro studies of transcription frequently require the preparation of defined elongation complexes. Defined transcription elongation complexes (TECs) are typically prepared by constructing an artificial transcription bubble from synthetic oligonucleotides and RNA polymerase. This approach is optimal for diverse applications but is sensitive to nucleic acid length and sequence and is not compatible with systems where promoter-directed initiation or extensive transcription elongation is crucial. To complement scaffold-directed approaches for TEC assembly, I have developed a method for preparing promoter-initiated Escherichia coli TECs using a purification strategy called selective photoelution. This approach combines TEC-dependent sequestration of a biotin–triethylene glycol transcription stall site with photoreversible DNA immobilization to enrich TECs from an in vitro transcription reaction. I show that selective photoelution can be used to purify TECs that contain a 273-bp DNA template and 194-nt structured RNA. Selective photoelution is a straightforward and robust procedure that, in the systems assessed here, generates precisely positioned TECs with >95% purity and >30% yield. TECs prepared by selective photoelution can contain complex nucleic acid sequences and will therefore likely be useful for investigating RNA structure and function in the context of RNA polymerases.

A novel assay strategy based on isothermal amplification and cascade signal amplified electrochemical DNA sensor for sensitive detection of Helicobacter pylori

A novel and sensitive Helicobacter pylori assay strategy based on linear isothermal amplification reaction (LIAR) and DNAzymes catalyzed DNA sensor was developed. A 17 nt target DNA (tDNA) specific in the H. pylori genome was selected and used as primer in the LIAR. A template DNA was designed to produce an amplicon with Mg2+-dependent and RNA-cleaving DNAzyme (Md-Dz) activity. The amplicon (i.e. Md-Dz) was then detected with the DNA sensor. The DNA sensor was fabricated simply through immobilization of a hairpin DNA that contains both Md-Dz substrate and G-quadruplex DNAzyme (Gq-Dz), on gold electrodes. The Gq-Dz possesses peroxidase activity, catalyzing the electro-redox of the electrochemical signal probe hydroquinon. The LIAR, Md-Dz catalytic reaction, and DNA sensor fabrication and detection processes were characterized and optimized. The LIAR amplification efficiency is as high as ~5 × 105 fold, and the assay strategy is very sensitive, selective and repetitive. The differential pulse voltammetry (DPV) detection signals linearly correlate to the logarithmic tDNA concentration in the range of 0.1–10,000 fM, with a limit of detection (LOD) of 34 aM. The detection linear range for H. pylori genomic DNA is 2.1–67.2 pg, with an LOD of 1.3 pg. Muted DNA sequences and other pathogenic bacteria did not result in interferences. In addition, the DNA sensor is very stable in storage. More importantly, the DNA sensor and the assay strategy could be universally applicable to detect any biological sample and short DNA sequence of interests.

Preparation of a poly (PEGDA‐co‐GMA) thin hydrogel matrix for oligonucleotide microarray applications

AbstractBACKGROUNDFabricating oligonucleotide microarrays with high immobilization efficiency and sensitivity by a simple surface modification route remains highly desirable in the biochip field. Here we develop a facile method to prepare a 3D crosslinking thin hydrogel film on glass slides for high‐density immobilization of DNA probes.RESULTSThin hydrogel film using poly (ethylene glycol) diacrylate (PEGDA) as crosslinker and glycidyl methacrylate (GMA) as functional monomer was attached on vinyl group‐modified glass slides by a photopolymerization technique. The effect of monomer ratios (PEGDA:GMA) on the surface hydrophilicity of the hydrogel layer and the immobilization density of DNA probes were investigated and an optimal monomer ratio of 10:1 and te maximum immobilization density of 1.4 pmol cm–2 were found. Adjusting the hydrophilicity of the hydrogel film effectively suppressed its swelling and produced a typical dense but not porous structure with a flat surface favorable for printing microarrays. The maximum immobilization density and efficiency of oligonucleotide probes on the 3D hydrogel film were 1.4 pmol cm–2 and 83%, respectively, which are superior values to those on a 1D support. The microarray had a wide detection range of target nucleotides from 15.3 pmol L–1 to 62.5 nmol L–1. In addition, the hybridization assay demonstrated that the microarray showed high sensitivity with a limit of detection of 30.5 pmol L–1.CONCLUSIONThe poly (PEGDA‐co‐GMA) hydrogel film could be grafted on the glass slide by the photopolymerization technique and the DNA probe was effectively immobilized on the thin hydrogel by reacting with the epoxy groups. This strategy of attaching a thin cross‐linked poly(PEGDA‐co‐GMA) hydrogel layer on glass slides has promising prospects in the fields of bioanalysis and microarrays. © 2021 Society of Chemical Industry (SCI).

Laser-scribed graphene nanofiber decorated with oil palm lignin capped silver nanoparticles: a green biosensor

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), requires a high level of attention and is one of the most infectious diseases in the air. Present methods of diagnosing TB remain ineffective owing to their low sensitivity and time consumption. In this study, we produced a green graphene nanofiber laser biosensor (LSG-NF) decorated with oil palm lignin-based synthetic silver nanoparticles (AgNPs). The resulting composite morphology was observed by field-emission scanning electron microscopy and transmission electron microscopy, which revealed the effective adaptation of the AgNPs to the LSG-NF surface. The successful attachment of AgNPs and LSG-NFs was also evident from X-ray diffraction and Raman spectroscopy studies. In order to verify the sensing efficiency, a selective DNA sample captured on AgNPs was investigated for specific binding with M.tb target DNA through selective hybridisation and mismatch analysis. Electrochemical impedance studies further confirmed sensitive detection of up to 1 fM, where a detection limit of 10−15 M was obtained by estimating the signal-to-noise ratio (S/N = 3:1) as 3σ. Successful DNA immobilisation and hybridisation was confirmed by the detection of phosphorus and nitrogen peaks based on X-ray photoelectron spectroscopy and Fourier-transform infrared spectroscopy. The stability and repeatability of the analysis were high. This approach provides an affordable potential sensing system for the determination of M. tuberculosis biomarker and thus provides a new direction in medical diagnosis.

Oriented Soft DNA Curtains for Single-Molecule Imaging.

Over the past 20 years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes a flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using a protein template-directed assembly. In our assay, a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended by either single- or both-end immobilization to the protein templates. For oriented both-end DNA immobilization, we employed heterologous DNA labeling and protein template coverage with the antidigoxigenin antibody. In contrast to single-end immobilization, both-end immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein–DNA interactions at more controllable reaction conditions. Additionally, we increased the immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g., CRISPR-Cas9) binding assay that monitors the binding location and position of individual fluorescently labeled proteins on DNA.

Cyclic voltammetry readout circuitry for DNA biosensor application

Cyclic voltammetry electrochemical biosensors reported a wide usage and applications for its fast response, able to be miniaturized and its sensitivity. However, the bulky, expensive and laboratory-based readout circuitry made it impossible to be used in the field-based environment. A miniaturized and portable readout circuitry for the DNA detection using hybridization technique had been design and developed in this work. It embedded with fabricated FR4 based sensor and produced respective current when the applied voltage was within the range of 0 to 0.5 V. The readout circuitry had been verified with five analysis environments. Bare Au with distilled water (dH2O), bare Au with ferricyanide reagent solution, DNA immobilization, DNA non-hybridization and DNA hybridization. All the results performed produced peak cathodic current when the applied input voltage is within 0.5 V to 3 V and hence proved that the miniaturized and portable readout circuitry is suitable to be implemented for cyclic voltammetry electrochemical biosensor.

DNA immobilization and detection using DNAbinding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNAbinding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNAbinding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNAbinding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNAbinding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNAbinding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.