Electrochemical DNA Detection Research Articles

Synthesis and characterization of polysaccharide-cryogel and its application to the electrochemical detection of DNA

The main goal of our study is to demonstrate the applicability of the PPy-cryogel-modified electrodes for electrochemical detection of DNA. First, a polysaccharide-based cryogel was synthesized. This cryogel was then used as a template for chemical polypyrrole synthesis. This prepared polysaccharide-based conductive cryogel was used for electrochemical biosensing on DNA. Carrageenan (CG) and sodium alginate (SA) polysaccharides, which stand out as biocompatible materials, were used in cryogel synthesis. Electron transfer was accelerated by polypyrrole (PPy) synthesized in cryogel networks. A 2B pencil graphite electrode with a diameter of 2.00 mm was used as a working electrode. The prepared polysaccharide solution was dropped onto a working electrode as a support material to improve the immobilization capacity of biomolecules and frozen to complete the cryogelation step. PPy synthesis was performed on the electrodes whose cryogelation process was completed. In addition, the structures of cryogels synthesized on the electrode surface were characterized by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Surface characterization of the modified electrodes was performed by energy-dispersive X-ray spectroscopy (EDX) analysis. Electrochemical determination of fish sperm DNA (fsDNA) was performed using a PPy-cryogel-modified electrode. The use of a porous 3D cryogel intermediate material enhanced the signal by providing a large surface area for the synthesis of PPy and increasing the biomolecule immobilization capacity. The detection limit was 0.98 µg mL−1 in the fsDNA concentration range 2.5–20 µg mL−1. The sensitivity of the DNA biosensor was estimated to 14.8 µA mM−1 cm−2. The stability of the biosensor under certain storage conditions was examined and observed to remain 66.95% up to 45 days.Graphical

Polymerase incorporation of 4-nitrophenyl modified 2′-deoxyuridine-5′-triphosphates into double-stranded DNA for direct electrochemical detection

Three novel 2′-deoxyuridine-5′-triphosphates modified with 4-nitrophenyl groups via various linkers (dUTP-N1, dUTP-N2, and dUTP-N3) were tested as bearers of reducible electroactive labels as well as substrates suitable for enzymes used in polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) with a potential application to direct electrochemical detection of double-stranded deoxyribonucleic acid (dsDNA). In cyclic and square wave voltammograms on carbon screen printed electrodes, the labeled dUTP have demonstrated distinct reduction peaks at potentials of –0.7 V to –0.9 V (phosphate buffer, pH 7.4). The reduction peak currents of dUTP-N derivatives were found to increase with their molar concentrations. The dUTP-N3 with a double bond in the linker had the lowest reduction potential (about 100 mV less negative) among the derivatives studied. Further, dUTP-N nucleotides were tested as substrates in PCR and RPA to incorporate the electroactive labels into 90, 210, or 206 base pair long dsDNA amplicons. However, only a dUTP-N1 derivative with a shorter linker without the double bond demonstrated satisfactory compatibility with both PCR and RPA, though with a low reaction output of modified dsDNA amplicons (at 100% substitution of dTTP). The dsDNA amplicons produced by PCR with 85% substitution of dTTP by the dUTP-N1 in the reaction mixture were successfully detected by square wave voltammetry at micromolar concentrations at high square wave frequency.

Synergistic signal amplification of a 3D Dual-Core DNA nanomachine and PCNs@AuPdCe hybrid nanozymes for ultrasensitive electrochemical detection of Cell-free DNA

The potential for early cancer detection through cell-free DNA (cfDNA) analysis is hindered by the low abundance and susceptibility to chemical damage of cfDNA, presenting a formidable challenge for precision analysis. In this study, we introduce an innovative electrochemical biosensor that leverages a three-dimensional (3D) dual-core DNA nanomachine driven by exonuclease III (EXO III) and trimetallic-deposited porous coordination network-224 hybrid nanozymes (PCNs@AuPdCe). The DNA nanomachine is designed with target cfDNA and 8-legged polyamidoamine-ssDNA (octopus-like) as dual dynamic cores, while magnetic beads serve as the 3D orbit, enabling highly stable cascade amplification from 1 to 8 to N. The newly synthesized PCNs@AuPdCe exhibits remarkable superoxide dismutase-, catalase-, and peroxidase-like activity and serves as a signal tag. The synergistic amplification resulting from the dual-core DNA nanomachine and PCNs@AuPdCe enables the biosensor to detect cfDNA levels as low as “a” M with a broad detection range from 1 fM to 1 nM. Analysis of real plasma samples demonstrates the robust anti-interference ability and promising clinical applications of the biosensor. Therefore, the construction of this electrochemical biosensor is of great significance to promote the development of in vitro diagnostic technology for early cancer screening.

Polymerase incorporation of fluorescein or rhodamine modified 2′-deoxyuridine-5′-triphosphates into double-stranded DNA for direct electrochemical detection

The 2′-deoxyuridine-5′-triphosphates modified with fluorescein (dUTP-Fl) or rhodamine (dUTP-Rh) were tested as bearers of electroactive labels and as proper substrates for polymerases used in polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) with the aim of electrochemical detection of double-stranded DNA (dsDNA) amplification products. For this purpose, electrochemical behavior of free fluorescein and rhodamine as well as the modified nucleotides, dUTP-Fl and dUTP-Rh, was studied by cyclic (CV) and square wave (SWV) voltammetry on carbon screen printed electrodes. Both free fluorescein and dUTP-Fl underwent a two-step oxidation at the peak potentials (Ep) of 0.6–0.7 V and 0.8–0.9 V (phosphate buffer, pH 7.4). The reduction peaks of fluorescein and dUTP-Fl were registered between –0.9 V and −1 V, but they did not depend on concentration. The free rhodamine and dUTP-Rh have demonstrated the well-defined oxidation peaks at 0.8–0.9 V. In addition, the distinct reduction peaks at Ep between –0.8 V and −0.9 V were registered for both rhodamine and dUTP-Rh. The dUTP-Fl and dUTP-Rh were further tested as substrates to incorporate an electroactive label into 210 or 206 base pair long dsDNA amplicons generated either by PCR or RPA. Among two dUTP derivatives tested, dUTP-Fl revealed significantly better compatibility with PCR and RPA, producing the full-size amplicons at 50–90% substitution of dTTP in the reaction mixture. In the PCR, the best compromise between amplicon output and labeling was achieved at the dUTP-Fl : dTTP and dUTP-Rh : dTTP molar ratios of 70% : 30% and 20% : 80% in the PCR mixture, respectively, allowing the direct electrochemical detection of amplicons at micromolar concentrations. Alongside with fluorescence DNA assays, the fluorescein and rhodamine modified dUTP appear as promising electroactive labels to develop direct electrochemical DNA assays for detecting PCR and RPA products.

Redox Indicator-Based Electrochemical DNA Detection

Redox Indicator-Based Electrochemical DNA Detection

CVD Graphene Electrode for Direct Electrochemical Detection of Double-Stranded DNA

Understanding and regulating DNA interactions with solvents and redox-active centers opens up new possibilities for improving electrochemical signals and developing adequate biosensors. This work reports the development of a modified indium tin oxide (ITO) electrode by chemical vapor deposition (CVD) of graphene for the detection of double-stranded DNA. The modified electrode shows a better electrical conductivity than ITO, as confirmed by electrochemical impedance spectroscopy (EIS), where a drastic decrease in the charge–transfer resistance, Rct, from ~320 to ~60 Ω was observed. Sequences of double-stranded genomic DNA with a different number of base pairs are evaluated through differential pulse voltammetry (DPV), using ferri/ferrocyanide ([Fe(CN)6]3−/4−) as a mediator in the solution. Variations in the electrochemical response of the [Fe(CN)6]3−/4− probe are observed after introducing redox inactive double-stranded DNA ions. The redox-active [Fe(CN)6]3−/4− probe serves as a scaffold to bring DNA into the graphene-modified ITO electrode surface, provoking an increase in the current and a change in the potential when the number of base pairs increases. These results are confirmed by EIS, which shows a variation in the Rct. The calibration of DPV intensity and Rct vs. DNA base pairs (bps) number were linear in the 495–607 bps range. The proposed method could replace the nucleic acid gel electrophoresis technique to determine the presence of a DNA fragment and quantify its size.

Label-free electrochemical detection of cancer biomarkers DNA and anti-p53 at tin oxide quantum dot-gold-DNA nanoparticle modified electrode

Lung cancer is one of the deadliest types of cancer and accounts for 8.1% of all cancer related deaths. To prevent a growing death rate, it is crucial to identify lung cancer at an early stage by single polynucleotide morphism detection. In this paper, we present a novel label-free electrochemical biosensor based on composites of tin oxide quantum dots and gold nanoparticles (SnO2-QD-Au) for the sensitive and precise detection of lung cancer DNA. The SnO2-QD and SnO2-QD-Au nanoparticles were characterized using Scanning and Transmission Electron Microscopes (SEM and TEM), X-ray diffractometer (XRD), X-ray photoelectron spectroscopy (XPS), UV–Vis spectroscopy (UV), Fourier transmission infrared spectroscopy (FTIR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Gold thiol covalent bonding was used for immobilising probe DNA on the surface of SnO2-QD-Au nanoparticles followed by target DNA hybridization and detected electrochemically in presence of 1 mM [Fe(CN6)]3−/4−as a redox couple probe. Under ideal circumstances, the sensor showed the lowest detection limit of 3.2 × 10−20 M with a linear range of 1 × 10−6 − 1 × 10−20 M. Additionally, the sensing method was applied to find a cancer biomarker, Anti-P53 antibody.

Electrochemical Detection of Femtomolar DNA via Boronate Affinity-Mediated Decoration of Polysaccharides with Electroactive Tags.

In view of their high efficiency and cost-effectiveness, polymers are of great promise as carriers for signal tags in amplified detection. Herein, we present a polysaccharide-amplified method for the electrochemical detection of a BRCA1 breast cancer gene-derived DNA target at the femtomolar levels. Briefly, peptide nucleic acid (PNA) with a complementary sequence was tethered as the capture probe for the DNA target, to which carboxyl group-containing polysaccharides were then attached via facile phosphate-Zr(IV)-carboxylate crosslinking, followed by the decoration of polysaccharide chains with electroactive ferrocene (Fc) signal tags via affinity coupling between a cis-diol site and phenylboronic acid (PBA) group. As the polysaccharide chain contains hundreds of cis-diol sites, boronate affinity can enable the site-specific decoration of each polysaccharide chain with hundreds of Fc signal tags, efficiently transducing each target capture event into the decoration of many Fc signal tags. As polysaccharides are cheap, renewable, ubiquitous, and biodegradable natural biopolymers, the use of polysaccharides for signal amplification offers the benefits of high efficiency, cost-effectiveness, excellent biocompatibility, and environmental friendliness. The linear range of the polysaccharide-amplified method for DNA detection was demonstrated to be from 10 fM to 10 nM (R2 = 0.996), with the detection limit as low as 2.9 fM. The results show that this method can also discriminate single base mismatch with satisfactory selectivity and can be applied to DNA detection in serum samples. In view of these merits, the polysaccharide-amplified PNA-based electrochemical method holds great promise in DNA detection with satisfactory sensitivity and selectivity.

Electrochemical and Optical Detection of Plant DNA for Sex Determination in a Lab-on-Chip Prototype

INTRODUCTION The fruit industry employs millions of hectares for production; of these, a significant percentage uses only female or hermaphrodite plants. The selection is carried out by morphological analysis until blooming, which represents an inefficient use of resources. Typically, well-equipped laboratories and trained personnel are required to perform DNA analysis and give valid results1,2. Devices capable of generating this information within a short timespan could contribute to a fast and effective selection of productive plants. Nonetheless, it is necessary to have high sensitivity to differentiate between samples. The objective of this work is the development of an electrochemical and an optical detection system, with high sensitivity and repeatability. With the aim to incorporate the best option in a fully integrated device that ensures an easy-to-use product, safeguarding the correct use by non-trained operators and reducing the time to obtain a result. MATERIALS AND METHODS To evaluate the detection systems a random sequence of DNA (5’-TAGTCGTCAATCCTCCGCTT) was used (50 – 540 ng/µL). The electrochemical detection design is composed by three electrodes (WE-Au, AE-Au, RE-Ag|AgCl) and 0.5 M Ru(NH3)6Cl3 as a DNA intercalating agent3 in 40 mM TAE buffer. Three electrochemical techniques (SWV, DPV and CV) were compared (Bio Logic potentiostat). The optical detection system is composed of a phototransistor (Max. Sens. @ 550 nm), a filter (520 nm) and a LED (Max. WL peak @ 455 nm) to detect the fluorescent DNA intercalator SYBRgreen. Different concentrations of fluorescein (0.005 – 10 µM) were used to evaluate the system capabilities. The system was evaluated with the DNA sequence and SYBRgreen, then compared with a fluorescence microscope (Nikon Eclipse Ti-U). Finally, both systems were tested with plant DNA samples. Genomic material was extracted (CTAB protocol4) form 6 plants (3 female/3 hermaphrodite), amplified by PCR (Thermal cycler miniPCR Amplyus) and analyzed by electrophoresis. RESULTS With the electrochemical detection system, we observed a proportional decrease in the Ru(NH3)6Cl3 signal as the DNA concentration increases, as expected. The data analysis demonstrated that SWV was the most sensitive technique with a R2=0.980, LOD 30.968 ng/µL and LOQ 103.226 ng/µL. Results showed that female plants present an average current of 1.774 μA while hermaphrodite plants had 1.694 μA, allowing us to differentiate hermaphrodite plants accurately with a 90 % confidence interval. On the other hand, in the optical detection system with a R2=0.9568, LOD 2.12 ng/µL and LOQ 7.06 ng/µL, we observed an increase in the signal as de DNA concentration increases. When the optical analysis was compared with the fluorescence microscope results, we observed a similar trend in the signals with a R2=0.995, that validate the functionality of our system. However, the difference between plant samples was detected with only the 70 % confidence interval, female samples showed an average signal of 381 mV while hermaphrodite plants had 352.56 mV. CONCLUSIONS Plant DNA detection was evaluated through two different systems. The optical detection system showed more sensitivity with synthetic samples. However, in the evaluation of plant samples, the electrochemical system is less susceptible to interferences. Therefore, we can differentiate between female and hermaphrodite plants with a 90 % confidence interval. In this way, it represents the best option to evaluate a lab-on-a-chip device. REFERENCES Barrantes-Santamaría W, Loría-Quirós C, Gómez-Alpízar L. Evaluation of two-sex determining systems in papaya plants (Carica papaya) Pococí hybrid. Agron Mesoamerican. 2019;30(2):437-446. doi:10.15517/am.v30i1.34916Cornelis S. FORENSIC LAB-ON-A-CHIP DNA ANALYSIS. 2019.Li LY, Jia HN, Yu HJ, et al. Synthesis, characterization, and DNA-binding studies of ruthenium complexes [Ru(tpy)(ptn)] 2 + and Ru(dmtpy)(ptn)] 2 +. J Inorg Biochem. 2012;113:31-39. doi:10.1016/j.jinorgbio.2012.03.008Biosciences. CTAB extraction solution for genomic DNA extraction. Biosciences. 2016:3-7. Figure 1

Hemoglobin-catalyzed atom transfer radical polymerization for ultrasensitive electrochemical DNA detection

The use of hemoglobin (Hb) to drive atom transfer radical polymerization (ATRP) process (Hb-ATRP) for detection of lung cancer related nucleic acid is firstly reported. Hb does not need to be treated prior to using indicating the potential for synthetic engineering in complex biological microenvironments without the need for in vitro techniques. Here, we report a new signal amplification strategy using Hb-mediated graft of nitronyl niroxide monoradical polymers as a signal-on electrochemical biosensor for ultralow level DNA highly selective detection. Building DNA biosensors includes: (i) the fixation of peptide nucleic acid (PNA) probe (no phosphate group) via the 5′ terminus-SH; (ii) the modification of transition metal; (iii) Site-specific markers of Hb-ATRP promoter, and (iv) the grafting of polymers with electrochemical signal by Hb-ATRP process. Through the Hb-ATRP process of nitronyl nitroxide monoradical (TEMPO), the presence of a small amount of DNA can eventually result in calling a certain number of TEMPO redox tags. Obviously, the Hb-ATRP is a method of easy source of raw materials, simple operation and no need for complex equipment. The constructed biosensor, as expected, is highly selective and sensitive to target DNA. The detection limit can be calculated as 15.96 fM under optimal conditions. The excellent performance also shows that the constructed DNA biosensor is suitable for DNA screening and DNA concentration determination in complex sample matrix.

Nanocomposite of electrochemically reduced graphene oxide and gold nanourchins for electrochemical DNA detection

A nanocomposite of graphene oxide and gold nanourchins has been used here to modify the surface of a screen‐printed carbon electrode to enhance the sensitivity of the electrochemical DNA detection system. A specific single‐stranded DNA probe was designed based on the target DNA sequence and was thiolated to be self‐assembled on the surface of the gold nanourchins placed on the modified electrode. Doxorubicin was used as an electrochemical label to detect the DNA hybridisation using differential pulse voltammetry (DPV). The assembling process was confirmed using scanning electron microscopy (SEM) imaging, cyclic voltammetry (CV), and the EIS method. The high sensitivity of the proposed system led to a low detection limit of 0.16 fM and a wide linear range from 0.5 to 950.0 fM. The specificity of the DNA hybridisation and the signalling molecule (haematoxylin) caused very high selectivity towards the target DNA than other non‐specific sequences.

Au Nanoparticle-Based Amplified DNA Detection on Poly-l-lysine Monolayer-Functionalized Electrodes.

Affinity sensing of nucleic acids is among the most investigated areas in biosensing due to the growing importance of DNA diagnostics in healthcare research and clinical applications. Here, we report a simple electrochemical DNA detection layer, based on poly-l-lysine (PLL), in combination with gold nanoparticles (AuNPs) as a signal amplifier. The layer shows excellent reduction of non-specific binding and thereby high contrast between amplified and non-amplified signals with functionalized AuNPs; the relative change in current was 10-fold compared to the non-amplified signal. The present work may provide a general method for the detection of tumor markers based on electrochemical DNA sensing.

Coenzyme−catalyzed electroinitiated reversible addition fragmentation chain transfer polymerization for ultrasensitive electrochemical DNA detection

Ultrasensitive detection of biomarkers at an early stage is generally limited by external influence factors such as high reaction temperature, complex operations, and sophisticated instruments. Here, we circumvent these problems by using nicotinamide adenine dinucleotide (NAD+) to control electroinitiated reversible addition fragmentation chain transfer (electro−RAFT) polymerization for biosensing that enables the detection of a few molecules of target DNA. In this coenzyme−catalyzed electro−RAFT polymerization, numerous ferrocenylmethyl methacrylate (FCMMA) as monomer with electrochemistry signal were linked to the biomarker on Au electrode. Afterwards, a strong oxidation peak appears at the potential of about 0.3 V that represents a typical oxidation potential of FCMMA. The sensitivity of this methodology was presented by detecting DNA from 10−1 to 104 fM concentration and detection limit (LOD) being down to 4.39 aM in 10 μL samples. This is lower by factors than detection limits of most other ultra−sensitive electrochemical DNA assays.

Electric field-enhanced electrochemical CRISPR biosensor for DNA detection

DNA detection plays an important role in the rapid screening of cancers and early diagnosis of infectious diseases. Here, we developed a simple, versatile, electric field-enhanced (EFE), electrochemical CRISPR biosensor to detect DNA targets in a homogeneous solution phase. To improve the detection sensitivity, we applied a pulsed electric field to enrich nucleic acids on the electrode surface. The EFE electrochemical CRISPR biosensor takes advantage of the diffusivity difference between electrochemical oligonucleotide probes and CRISPR-cleaved probes toward a negatively charged working electrode, enabling simple and sensitive electrochemical detection of DNA without the need for complicated immobilization processing of electrochemical probes. Our developed CRISPR biosensor directly detects unamplified human papillomavirus-16 (HPV-16) DNA with a sensitivity of 1 pM. Further, the EFE electrochemical CRISPR biosensor coupled with recombinase polymerase amplification (RPA) successfully detects HPV-16 DNA in clinical samples. Thus, the EFE electrochemical CRISPR biosensor provides a simple, robust, and sensitive detection method for nucleic acid-based molecular diagnostics.

Electrochemical detection of DNA by formation of efficient electron transfer pathways through adsorbing gold nanoparticles to DNA modified electrodes

The high-performance detection of nucleic acids is increasingly important in the early and accurate disease diagnosis. Herein, we describe a unique label-free electrochemical DNA biosensor, which is based on gold nanoparticles (GNPs) mediated electron transfer through a DNA monolayer modified gold electrode surface. We observed that the different states of DNA monolayers (single-strand DNA or triple-helix DNA) and the different sizes of GNPs have a significant effect on the electron transfer by the DNA modified gold electrodes. This DNA biosensing strategy avoids the complicated fabrication process of the DNA capture probes and minimizes the steps of electrode pre-treatment. Under optimal conditions, this method can sensitively and accurately detect the target DNA ranging from 10 pM to 100 nM with a detection limit of 1.47 pM.

Highly Accurate and Fast Electrochemical Detection of Scrub Typhus DNA via a Nanoflower NiFe-Based Biosensor.

Owing to the lack of specific diagnostic methods, Scrub typhus can sometimes be difficult to diagnose in the Asia-Pacific region. Therefore, an efficient and rapid detection method urgently needs to be developed. Based on competitive single-stranded DNA over modified glassy carbon electrode (GCE), an electrochemical biosensor was established to detect the disease. The nano-flower NiFe layered double hydroxide (NiFe-LDH) modified GCE has a large specific surface area, which supported a large amount of gold nanoparticles, so that a wide linear detection range from 25 fM to 0.5 μM was obtained. The beacon DNA (B-DNA) with the same sequence as the Scrub typhus DNA (T-DNA), but labeled with methylene blue, was used to construct a competitive relationship. When T-DNA and B-DNA were present on the sensor simultaneously, they would hybridize with probe DNA in a strong competition, and the corresponding electrochemical response signal would be generated via testing. It contributed to reducing tedious experimental procedures and excessive response time, and achieved fast electrochemical detection of DNA. The strategy provides a worthy avenue and possesses promising applications in disease diagnosis.

An integrated E-Tube cap for sample preparation, isothermal amplification and label-free electrochemical detection of DNA

A simple, disposable, and integrated electronic-tube cap (E-tube cap) for DNA detection at the point-of-care was designed, fabricated, and tested. The E-tube cap contains a 3D printed electrode substrate for DNA extraction and label-free pH sensing detection. One Flinders Technology Associates (Whatman FTA) membrane was incorporated into the 3D printed electrode substrate for the isolation, concentration, and purification of DNA. The E-tube cap with captured DNA by the membrane was inserted directly into a reaction tube for loop-mediated isothermal amplification (LAMP). The isothermal amplification process was monitored in real-time by a 3D printed electrochemical electrode coated with pH-sensitive material (carbon/iridium oxide layer). The pH sensing electrode showed an excellent linear response within the pH range of 6–9 with a slope of −31.32 ± 0.5 mV/pH at room temperature. The utility of the integrated E-tube cap was demonstrated by detecting the presence of lambda DNA spiked in saliva samples with a sensitivity of 100 copies per mL sample within 30 min. Such a simple, rapid, and affordable diagnostic device is particularly suitable for point-of-care molecular diagnostics of infectious diseases.

Ultrasensitive electrochemical detection of miRNA coupling tetrahedral DNA modified gold nanoparticles tags and catalyzed hairpin assembly

MicroRNAs (miRNAs) play key regulatory roles in a number of biological processes, which act as critical biomarkers for clinical diagnosis. There are urgent needs to develop advanced tools for accurate and convenient analysis of miRNA in biological circumstances. In this study, an ultrasensitive electrochemical biosensor for miRNA assay is fabricated. Tetrahedral DNA modified gold nanoparticles tags are applied with optimized orientation, which are able to recruit a large number of electrochemical species for remarkable signal responses. Benefiting from the excellent amplification efficiency of the association of strand displacement amplification and catalyzed hairpin assembly, the established method shows ultrahigh sensitivity with the limit of detection as low as 10 aM. A wide linear range from 10−17 to 10−7 M is achieved. In addition, this method is capable to specifically discriminate interfering miRNAs with slightly different sequences. The successful assessment of miRNA levels in human serum samples also demonstrates good practical utility. Therefore, the proposed method has great potential to the applications of miRNA expression profiling and biological studies.

DNA electrochemical biosensor for detection of Alicyclobacillus acidoterrestris utilizing Hoechst 33258 as indicator

Alicyclobacillus acidoterrestris is an acidophilic and thermophilic bacterium present in the soil, often associated with the spoilage of acidic juices, such as orange juice. Their spores resist pasteurization and, when reactivated, modify the organoleptic properties of the juice, making it unsuitable for consumption, due mainly to production of guaiacol. Biosensors are detection devices that respond quickly and are easy to handle, with great potential for use in the juice production chain. In this context, this work reports an electrochemical genosensor for detection of A. acidoterrestris, based on a graphite electrode modified with electrochemically reduced graphene oxide, a polymer derived from 3-hydroxybenzoic acid and a specific DNA probe sequence complementary with the genomic DNA of A. acidoterrestris. Detection of the target was performed by monitoring the oxidation peak of the Hoechst 33258, a common DNA stainer. The genosensor detection limit was 12 ng mL−1 and it kept 77% of response after ten weeks, and a test showed that orange juice does not interfere with bacteria lysate detection. This biosensor is the first platform for electrochemical detection of the genomic DNA of A. acidoterrestris in the literature, and the first to use Hoechst 33258 as indicator with whole genomic DNA molecules.

Advanced electrochemical detection of nitrogenous bases, synthetic oligonucleotides, and single-stranded DNA through flow injection analysis and catalytic oxidation on Prussian Blue

The effect of Prussian Blue (PB, ferric hexacyanoferrate) on oxidation of free nucleobases, synthetic oligonucleotides, single- and double-stranded DNA (ssDNA and dsDNA) was evaluated by cyclic voltammetry (CV) and flow injection analysis (FIA) on carbon screen printed electrodes, both bare (SPE) and PB modified (SPE/PB). It has been found that electrocatalytic oxidation of nucleobases, namely guanine, adenine, thymine, and cytosine, or nucleobase residues takes place via electrochemically generated Berlin Green (BG), fully oxidized form of PB. The constant potential FIA allowed one to register electrooxidation of all DNA nitrogenous bases at 0.95 V and three nitrogenous bases (except for cytosine) at 0.70 V on SPE and SPE/PB. The modification of electrode surface with PB resulted in a shift of oxidation potential to less positive values and enhanced (up to 8 times) detection sensitivity for DNA nucleobases. The pronounced catalytic effect of electrogenerated BG on their oxidation has also been observed for synthetic oligonucleotides and degraded ssDNA of natural origin. By fractionating ssDNA, it has been found that the main contributor to the total oxidation signal is the fraction of lowest molecular weight (< 3 kDa). At the same time, no oxidation signal was observed for large molecular weight native dsDNA in the potential range from 0 to 1 V on bare SPE and SPE/PB in both CV and FIA measurement modes. Therefore, the advanced electrochemical detection by FIA with SPE/PB was achieved for all nucleobases, synthetic oligonucleotides, and ssDNA of natural origin. Moreover, the obtained results expand the contemporary understanding of DNA electrochemistry.