Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification

The development and characterization of a magnetic bead (MB)-quantum dot (QD) nanoparticles based assay capable of quantifying pathogenic bacteria is presented here. The MB-QD assay operates by having a capturing probe DNA selectively linked to the signaling probe DNA via the target genomic DNA (gDNA) during DNA hybridization. The signaling probe DNA is labeled with fluorescent QD₅₆₅ which serves as a reporter. The capturing probe DNA is conjugated simultaneously to a MB and another QD₆₅₅, which serve as a carrier and an internal standard, respectively. Successfully captured target gDNA is separated using a magnetic field and is quantified via a spectrofluorometer. The use of QDs (i.e., QD₅₆₅/QD₆₅₅) as both a fluorescence label and an internal standard increased the sensitivity of the assay. The passivation effect and the molar ratio between QD and DNA were optimized. The MB-QD assay demonstrated a detection limit of 890 zeptomolar (i.e., 10⁻²¹ mol L⁻¹) concentration for the linear single stranded DNA (ssDNA). It also demonstrated a detection limit of 87 gene copies for double stranded DNA (dsDNA) eaeA gene extracted from pure Escherichia coli (E. coli) O157:H7 culture. Its corresponding dynamic range, sensitivity, and selectivity were also presented. Finally, the bacterial gDNA of E. coli O157:H7 was used to highlight the MB-QD assay's ability to detect below the minimum infective dose (i.e., 100 organisms) of E. coli O157:H7 in water environment.     

Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor for Escherichia coli detection

A Fe₂O₃@Au core/shell nanoparticle-based electrochemical DNA biosensor was developed for the amperometric detection of Escherichia coli (E. coli). Magnetic Fe₂O₃@Au nanoparticles were prepared by reducing HAuCl₄ on the surfaces of Fe₂O₃ nanoparticles. This DNA biosensor is based on a sandwich detection strategy, which involves capture probe immobilized on magnetic nanoparticles (MNPs), target and reporter probe labeled with horseradish peroxidase (HRP). Once magnetic field was added, these sandwich complexes were magnetically separated and HRP confined at the surfaces of MNPs could catalyze the enzyme substrate and generate electrochemical signals. The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 500 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be lowered to 5 cfu/mL of E. coli after 4.0 h of incubation.     

Nanostructured rough gold electrodes as platforms to enhance the sensitivity of electrochemical genosensors

An electrochemical DNA genosensor constructed by using rough gold as electrode support is reported in this work. The electrode surface nanopatterning was accomplished by repetitive square-wave perturbing potential (RSWPP). A synthetic 25-mer DNA capture probe, modified at the 5′ end with a hexaalkylthiol, able to hybridize with a specific sequence of lacZ gene from the Enterobacteriaceae bacterial family was assembled to the rough gold surface. A 25 bases synthetic sequence fully complementary to the thiolated DNA capture probe and a 326 bases fragment of lacZ containing a fully matched sequence with the capture probe, which was amplified by a specific asymmetric polymerase chain reaction (aPCR), were employed as target sequences. The hybridization event was electrochemically monitored by using two different indicators, hexaammineruthenium (III) chloride showing an electrostatic DNA binding mode, and pentaamineruthenium-[3-(2-phenanthren-9-yl-vinyl)-pyridine] (in brief RuL) which binds to double stranded DNA (dsDNA) following an intercalative mechanism. After optimization of the different variables involved in the hybridization and detection reactions, detection limits of 5.30 pg μL⁻¹ and 10 pg μL⁻¹ were obtained for the 25-mer synthetic target DNA and the aPCR amplicon, respectively. A RSD value of 6% was obtained for measurements carried out with 3 different genosensors prepared in the same manner.     

A sensitive DNA biosensor based on a facile sulfamide coupling reaction for capture probe immobilization

A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction. First, the versatile sulfonic dye molecule of 1-amino-2-naphthol-4-sulfonate (AN-SO₃⁻) was electrodeposited on the surface of a glassy carbon electrode (GCE) to form a steady and ordered AN-SO₃⁻ layer. Then the amino-terminated capture probe was covalently grafted to the surface of SO₃⁻-AN deposited GCE through the sulfamide coupling reaction between the amino groups in the probe DNA and the sulfonic groups in the AN-SO₃⁻. The step-by-step modification process was characterized by electrochemistry and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Using Ru(NH₃)₆³⁺ as probe, the probe density and the hybridization efficiency of the biosensor were determined to be 3.18 × 10¹³ strands cm⁻² and 86.5%, respectively. The hybridization performance of the biosensor was examined by differential pulse voltammetry using Co(phen)₃^3+/2+ (phen = 1,10-phenanthroline) as the indicator. The selectivity experiments showed that the biosensor presented distinguishable response after hybridization with the three-base mismatched, non-complementary and complementary sequences. Under the optimal conditions, the oxidation peak currents of Co(phen)₃^3+/2+ increased linearly with the logarithm values of the concentration of the complementary sequences in the range from 1.0 × 10⁻¹³ M to 1.0 × 10⁻⁸ M with a regression coefficient of 0.9961. The detection limit was estimated to be 7.2 × 10⁻¹⁴ M based on 3σ.     

Nucleic acid biosensor for detection of hepatitis B virus using 2,9-dimethyl-1,10-phenanthroline copper complex as electrochemical indicator

In this study, an electrochemical DNA biosensor was developed based on the recognition of target DNA by hybridization detection. The study was carried out using glassy carbon electrode (GCE) modified with lable-free 21-mer single-stranded oligonucleotides related to hepatitis B virus sequence via covalent immobilization and [Cu(dmp)(H₂O)Cl₂] (dmp = 2,9-dimethyl-1,10-phenanthroline) as an electrochemical indicator, whose sizes are comparable to those of the small groove of native double-duplex DNA. The method, which is simple and low cost, allows the accumulation of copper complex within the DNA layer. Electochemical detection was performed by cyclic voltammetry and differential pulse voltammetry over the potential range where the [Cu(dmp)(H₂O)Cl₂] was active. Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed the assay time. With this approach, a sequence of the hepatitis B virus could be quantified over the ranges from 8.82 × 10⁻⁸ to 8.82 × 10⁻⁷ M with a linear correlation of r = 0.9937 and a detection limit of 7.0 × 10⁻⁸ M. The [Cu(dmp)(H₂O)Cl₂] signal observed from probe sequence before and after hybridization with four bases mismatch containing sequence is lower than that observed after hybridization with complementary sequence.     

An easily-automated assay for the physiological state quantification of Pseudomonas sp. M18

In order to foreknow poorly performing cultures before wasting energy to scale them to large cultures, industrial microbial fermentation can greatly benefit from knowledge of the physiological state of cells. The method currently proposed is an easily automated physiological state determination method. We have designed one universal rRNA-specific probe for bacteria and developed novel signal probe hybridization (SPH) assay featuring no RNA extraction and no PCR amplification steps necessary to quantify the physiological state of microbial cells. The microbial cell was lysed with sonication and SDS. Signal probes were applied to hybridize and protect the rRNA target. S1 nuclease was then applied to remove the excessive signal probes, the single-stranded RNA and the mismatch RNA/DNA hybrids. The remaining signal probe was captured with a corresponding capture probe immobilized on a microplate and quantified with a horseradish peroxidase-conjugated color reaction. We then systemically optimized our assay. Results showed that the cell limit of detection (LOD) and the cell limit of quantification (LOQ) were 2.64 × 10⁴ cells and 9.86 × 10⁴ cells per well of microplate, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) of signal probe were 49.0 fM and 344.0 fM respectively. Using this technique, we quantified the 16S rRNA levels during the fermentation process of Pseudomonas sp. M18. Our results indicate that the 16S rRNA levels can directly inform us about the physiological state of microbial cells. This technique has great potential for application to the microbial fermentation industry.     

A reagentless DNA biosensor based on cathodic electrochemiluminescence at a C/CxO1−x electrode

A reagentless signal-on electrochemiluminescence (ECL) biosensor for DNA hybridization detection was developed based on the quenching effect of ferrocene (Fc) on intrinsic cathodic ECL at thin oxide covered glassy carbon (C/C_xO_1−x) electrodes. To construct the DNA biosensor, molecular beacon (MB) modified with ferrocene (3′-Fc) was attached to a C/C_xO_1−x electrode via the covalent bound between labeled amino (5′-NH₂) and surface functional groups. It was found that the immobilization of the probe on the electrode surface mainly depended on the fraction of surface carbonyl moiety. When a complementary target DNA (cDNA) was present, the stem-loop of MB on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the moving away of Fc from the electrode surface, and the restoring of the cathodic ECL signal. The restoration of the ECL intensity was linearly changed with the logarithm of cDNA concentration in the range of 1.0 × 10⁻¹¹ to 7.0 × 10⁻⁸ M, and the detection limit was ca. 5.0 pM (S/N = 3). Additionally, single-base mismatched DNA can be effectively discriminated from the cDNA. The great advantage of the biosensor lies in its simplicity and cost-effective with ECL generated from the electrode itself, and no adscititious luminophore is required.     

Electrochemiluminescence biosensor for the assay of small molecule and protein based on bifunctional aptamer and chemiluminescent functionalized gold nanoparticles

An electrochemiluminescence (ECL) biosensor for simultaneous detection of adenosine and thrombin in one sample based on bifunctional aptamer and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles (ABEI-AuNPs) was developed. A streptavidin coated gold nanoparticles modified electrode was utilized to immobilize biotinylated bifunctional aptamer (ATA), which consisted of adenosine and thrombin aptamer. The ATA performed as recognition element of capture probe. For adenosine detection, ABEI-AuNPs labeled hybridization probe with a partial complementary sequence of ATA reacted with ATA, leading to a strong ECL response of N-(aminobutyl)-N-(ethylisoluminol) enriched on ABEI-AuNPs. After recognition of adenosine, the hybridization probe was displaced by adenosine and ECL signal declined. The decrease of ECL signal was in proportion to the concentration of adenosine over the range of 5.0 × 10⁻¹²–5.0 × 10⁻⁹ M with a detection limit of 2.2 × 10⁻¹² M. For thrombin detection, thrombin was assembled on ATA modified electrode via aptamer–target recognition, another aptamer of thrombin tagged with ABEI-AuNPs was bounded to another reactive site of thrombin, producing ECL signals. The ECL intensity was linearly with the concentration of thrombin from 5 × 10⁻¹⁴ M to 5 × 10⁻¹⁰ M with a detection limit of 1.2 × 10⁻¹⁴ M. In the ECL biosensor, adenosine and thrombin can be detected when they coexisted in one sample and a multi-analytes assay was established. The sensitivity of the present biosensor is superior to most available aptasensors for adenosine and thrombin. The biosensor also showed good selectivity towards the targets. Being challenged in real plasma sample, the biosensor was confirmed to be a good prospect for multi-analytes assay of small molecules and proteins in biological samples.     

Molecular beacons: a real-time polymerase chain reaction assay for detecting Escherichia coli from fresh produce and water

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. The development of a real-time polymerase chain reaction (PCR) assay to detect the presence of Escherichia coli using these fluorogenic reporter molecules is reported. MBs were designed to recognize a 19-bp region of the uid A gene, coding for an enzyme β-glucuronidase. The specificity of the MB-based PCR assay was evaluated for various E. coli strains as well as bacteria species that are present in nature. The capability of the assay to detect E. coli in drinking water and produce was demonstrated. Positive detection of E. coli was demonstrated when >10¹ CFU mL⁻¹ (colony forming unit) was present in the water samples and fresh produce after 18 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli in food and environmental samples.     

Highly sensitive electrochemical impedance spectroscopic detection of DNA hybridization based on Aunano-CNT/PANnano films

A polyaniline nanofibers (PAN_nano)/carbon paste electrode (CPE) was prepared via dopping PAN_nano in the carbon paste. The nanogold (Au_nano) and carbon nanotubes (CNT) composite nanoparticles were bound on the surface of the PAN_nano/CPE. The immobilization and hybridization of the DNA probe on the Au_nano-CNT/PAN_nano films were investigated with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using methylene blue (MB) as indicator, and electrochemical impedance spectroscopy (EIS) using [Fe(CN)₆]^3−/4− as redox probe. The voltammetric peak currents of MB increased dramatically owing to the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films, and then decreased obviously owing to the hybridization of the DNA probe with the complementary single-stranded DNA (cDNA). The electron transfer resistance (R_et) of the electrode surface increased after the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films and rose further after the hybridization of the probe DNA. The remarkable difference between the R_et value at the DNA-immobilized electrode and that at the hybridized electrode could be used for the label-free EIS detection of the target DNA. The loading of the DNA probe on Au_nano-CNT/PAN_nano films was greatly enhanced and the sensitivity for the target DNA detection was markedly improved. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene and the polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from transgenically modified beans were determined with this label-free EIS DNA detection method. The dynamic range for detecting the PAT gene sequence was from 1.0 × 10⁻¹² mol/L to 1.0 × 10⁻⁶ mol/L with a detection limit of 5.6 × 10⁻¹³ mol/L.     

Electrochemiluminescence of CdTe quantum dots as labels at nanoporous gold leaf electrodes for ultrasensitive DNA analysis

A new electrochemiluminescence (ECL) DNA assay is developed using quantum dots (QDs) as DNA labels. When nanoporous gold leaf (NPGL) electrodes are used, sensitivity of the ECL assay is remarkably increased due to ultra-thin nanopores. In this assay, target DNA (t-DNA) is hybridized with capture DNA (c-DNA) bound on the NPGL electrode, which is fabricated by conjugating amino-modified c-DNA to thioglycolic acid (TGA) modified at the activated NPGL electrode. Following that, amino-modified probe DNA is hybridized with the t-DNA, yielding sandwich hybrids on the NPGL electrode. Then, mercaptopropionic acid-capped CdTe QDs are labeled to the amino group end of the sandwich hybrids. Finally, in the presence of S₂O₈²⁻ as coreactant, ECL emission of the QD-labeled DNA hybrids on the NPGL electrode is measured by scanning the potential from 0 to −2 V to record the curve of ECL intensity versus potential. The maximum ECL intensity (I_m,ECL) on the curve is proportional to t-DNA concentration with a linear range of 5 × 10⁻¹⁵ to 1 × 10⁻¹¹ mol/L. The ECL DNA assay can be used to determine DNA corresponding to mRNA in cell extracts in this study.     

Electrical detection of deoxyribonucleic acid hybridization based on carbon-nanotubes/nano zirconium dioxide/chitosan-modified electrodes

A novel and sensitive electrochemical DNA biosensor based on nanoparticles ZrO₂ and multi-walled carbon nanotubes (MWNTs) for DNA immobilization and enhanced hybridization detection is described. The MWNTs/nano ZrO₂/chitosan-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides were immobilized to the GCE. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using electroactive daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics increased DNA attachment quantity and complementary DNA detection sensitivity. The response signal increases linearly with the increase of the logarithm of the target DNA concentration in the range of 1.49 × 10⁻¹⁰ to 9.32 × 10⁻⁸ mol L⁻¹ with the detection limit of 7.5 × 10⁻¹¹ mol L⁻¹ (S/N = 3). The linear regression equation is I = 32.62 + 3.037 log C_DNA (mol L⁻¹) with a correlation coefficient value of 0.9842. This is the first application of carbon nanotubes combined with nano ZrO₂ to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization.     

Detection of thrombin using electrogenerated chemiluminescence based on Ru(bpy)3(2+)-doped silica nanoparticle aptasensor via target protein-induced strand displacement

A sensitive and selective aptasensor using tri(2,2′-bipyridyl)ruthenium(II)-doped silica nanoparticles (Ru(bpy)₃²⁺-doped SNPs) as DNA tags for detection of thrombin is developed based on the target protein-induced strand displacement of the DNA probe. For the proposed aptasensor, the aptamer was assembled on the surface of the Au electrode through Au-S binding. The hybridization event between the DNA probe labeled by the Ru(bpy)₃²⁺-doped SNPs and the aptamer was evaluated by electrogenerated chemiluminescence (ECL) measurements. Then, the DNA probe was displaced by thrombin and the binding event between the thrombin and the aptamer was monitored by ECL measurements again. The difference of ECL intensity (ΔI_ECL) of the two events could be used to quantify the thrombin. Other proteins, such as bovine serum albumin and bovine hemoglobin, had almost negligible ΔI_ECL. Under the optimal conditions, the ΔI_ECL was linearly related to the concentration of the thrombin in the range of 10 fM to 10 pM and the detection limit was down to 1.0 fM since SNPs containing a large number of Ru(bpy)₃²⁺ molecules were labeled on the DNA probe.     

Development and evaluation of a loop-mediated isothermal amplification method in conjunction with an enzyme-linked immunosorbent assay for specific detection of Salmonella serogroup D

Loop-mediated isothermal amplification in conjunction with enzyme-linked immunosorbent assay (LAMP-ELISA) provides a sensitive, specific and cost-effective method for detection of etiological causes of infections. The present study developed a reliable LAMP-ELISA diagnostic kit for identification of Salmonella serogroup D strains and evaluated its potential use in the detection of Salmonella serovars Enteritidis and Typhi. The LAMP-ELISA assay used a serogroup D/A-specific primer set to amplify a region of the prt gene, followed by hybridization of the digoxigenin-labeled products to a highly specific oligonucleotide probe for exact identification of serogroup D serovars. Among the bacteria tested, a positive reaction was only observed for strains belong to Salmonella serogroup D. The detection limit of the LAMP-ELISA assay was 4 CFU per tube, which was lower than PCR-ELISA assay with the same target gene (50 CFU per tube). Finally, the technique was successfully applied to an artificially contaminated meat sample with a detection limit 10³ CFU mL⁻¹, which was 10 times more sensitive than PCR-ELISA. Overall, the LAMP-ELISA assay could be used as a sensitive alternative method to PCR-ELISA for the specific detection of Salmonella serogroup D serovars in routine food microbiology or clinical laboratories worldwide.     

Electrochemical DNA biosensor for the detection of short DNA species of Chronic Myelogenous Leukemia by using methylene blue

A novel assay for the voltammetric detection of 18-bases DNA sequences relating to Chronic Myelogenous Leukemia (CML, Type b3a2) using methylene blue (MB) as the hybridization indicator was reported. DNA was covalently attached onto a glassy carbon electrode (GCE) through amines of the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N′-ethyl carbodiimidehydrochloride (EDC). The covalently immobilized single-stranded DNA (ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. A significant increase of the peak current for methylene blue upon the hybridization of immobilized ssDNA with cDNA in the solution was observed. This peak current change was used to monitor the recognition of CML DNA sequence. This electrochemical approach is sequence specific as indicated by the control experiments in which no peak current change was observed if a non-complementary DNA sequence was used. Factors, such as DNA target concentration and hybridization conditions determining the sensitivity of the electrochemical assay were investigated. Under optimal conditions, this sensor has a good calibration range between 1.25 × 10⁻⁷ and 6.75 × 10⁻⁷ M, with CML DNA sequence detection limit of 5.9 × 10⁻⁸ M.     

Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe

A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0 × 10² to 3.0 × 10⁴ cells mL⁻¹, with a detection limit of 2.6 × 10² cells mL⁻¹. The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL⁻¹. The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes.     

A sensitive DNA electrochemical biosensor based on magnetite with a glassy carbon electrode modified by muti-walled carbon nanotubes in polypyrrole

A novel sensitive electrochemical biosensor based on magnetite nanoparticle for monitoring DNA hybridization by using MWNT-COOH/ppy-modified glassy carbon electrode is described. In this new detection system, mercapatoacetic acid (RSH)-coated magnetite nanoparticles, capped with 5′-(NH₂) oligonucleotide, is used as DNA probe to complex 29-base polynucleotide target (a piece of human porphobilinogen deaminase PBGD promoter from 170 to 142). Target sequence hybridized with the probe results in the decrease of the reduction peak current of daunomycin connected with probe. The response of non-complementary sequence was almost the same as the blank, and the response of three-base mismatched sequence within 29-base polynucleotide was obviously distinguished from complementary sequence, which can easily identify point mutation of DNA. The equation of calibration plot is i_p (μA) = 0.8255 − 0.0847c_{target oligonucleotide} × 10¹³ in the range of 6.9 × 10⁻¹⁴ to 8.6 × 10⁻¹³ mol/L, and correlation coefficient is 0.9974. The detective limit is 2.3 × 10⁻¹⁴ mol/L of target oligonucleotide. This device can be optimized for the detection of complex sequence.     

Electrochemiluminescence DNA sensor based on Ru(bpy) 3 2+ -doped silica nanoparticle labeling and proximity-dependent surface hybridization assay

A new electrochemiluminescence (ECL) method based on the proximity-dependent surface hybridization assay and Ru(bpy)₃²⁺-doped silica nanoparticles (Ru-DSNPs) as labels were proposed for detecting DNA. The hybridization process involves two steps: firstly, the 3′ thiolated capture probe was self-assembled on the gold electrode. Secondly, the proximity-dependent surface hybridization assay was carried out. This proximity-dependent surface hybridization assay depended on the simultaneous recognition of a target DNA by a capture probe and Ru-DSNP-labeled probe and the formation of a duplex complex, which results in the luminophor approach to the electrode surface. Thus, sensitive ECL signals were obtained. Under optimum conditions, the intensity of the ECL of Ru-DSNPs was linearly related to the concentration of the target sequence in the range of 2.0 × 10⁻¹⁵ to 2.0 × 10⁻¹¹ mol/L. The detection limit was 1.0 × 10⁻¹⁵ mol/L (S/N = 3).     

Electrochemical detection of nicotinamide adenine dinucleotide based on molecular beacon-like DNA and E. coli DNA ligase

An electrochemical method for nicotinamide adenine dinucleotide (NAD⁺) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5′-SH and 3′-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD⁺, E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD⁺-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD⁺ range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD⁺ from its analogues.     

Time-resolved fluorescence based DNA detection using novel europium ternary complex doped silica nanoparticles

A two-probe tandem DNA hybridization assay including capture DNA₁, probe DNA₂, and target DNA₃ was prepared. The long-lived luminescent europium complex doped nanoparticles (NPs) were used as the biomarker. The complex included in the particle was Eu(TTA)₃(5-NH₂-phen)-IgG (ETN-IgG), the europium complex Eu(TTA)₃(5-NH₂-phen) linking an IgG molecule. Silica NPs containing ETN-IgG were prepared by the reverse microemulsion method, and were easy to label oligonucleotide for time-resolved fluorescence assays. The luminophores were well-protected from the environmental interference when they were doped inside the silica network. The sequences of Staphylococcus aureus and Escherichia coli genes were designed using software Primer Premier 5.0. Amino-modified capture DNA₁ was covalently immobilized on the common glass slides surface. The detection was done by monitoring the fluorescence intensity from the glass surface after the hybridization reaction with the NPs labeled probe DNA₂ and complementary target DNA₃. The sensing system presented short hybridization time, satisfactory stability, sensitivity, and selectivity. This approach was successfully employed for preliminary application in the detection of pure cultured E. coli, it might be an effective tool for pathogen DNA monitoring.