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.     

Amperometric detection of Enterobacteriaceae in river water by measuring β-galactosidase activity at interdigitated microelectrode arrays

Two simple methodologies are compared for the detection of faecal contamination in water using amperometry at gold interdigitated microelectrodes. They rely on the detection of β-galactosidase (β-gal) by redox cycling amperometry of the p-aminophenol (PAP) produced by the enzyme from the 4-aminophenyl β-d-galactopyranoside (PAPG) substrate. The use of phages as specific agents for the release of the bacteria-enclosed enzyme allowed the detection of 6 × 10⁵ CFU mL⁻¹Escherichia coli in 2 h without any pre-enrichment or preconcentration steps. Better limits of detection were achieved for the second strategy in the absence of phages. In this case, bacteria were enriched in the presence of both β-d-1-thiogalactopyranoside (IPTG) and substrate but in the absence of phages. Under such experimental conditions, 5 × 10⁴ CFU mL⁻¹E. coli could be detected after 2 h of incubation, while 7 h of incubation were enough to detect down to 10 CFU mL⁻¹ in river water samples. This represents a straightforward one-step method for the detection of faecal contamination that can be conducted in a single working day with minimal sample manipulation by the user.     

Analysis of Strychnos alkaloids in traditional Chinese medicines with improved sensitivity by sweeping micellar electrokinetic chromatography

A new amperometric method was developed for rapid detection of Escherichia coli (E. coli) density using a bienzyme biosensor. The bienzyme biosensor was fabricated based on the covalent immobilization of laccase and horseradish peroxidase (HRP) at indium tin oxide (ITO) electrode by (3-aminopropyl) triethoxysilane (APTES) monolayer. The bienzyme biosensor showed a high sensitivity in determination of the polyphenolic compounds, which was microbially generated from the salicylic acid (SA) added into the culture medium during the course of E. coli metabolism. Since the amount of polyphenolic compounds depends on E. coli density, the bienzyme biosensor was applied for the rapid and high sensitive detection of E. coli density after the E. coli solution was incubated in culture medium with salicylic acid for 2.5 h at 37 °C. By chronoamperometry, the amplified response current was obtained at the bienzyme biosensor, due to the substrate recycling of the polyphenolic compounds driven by bienzyme-catalyzed oxidation and electrochemical reduction. The amplified response current at the biosensor was linear with the E. coli density ranging from 1.6 × 10³ to 1.0 × 10⁷ cells/mL. The bienzyme biosensor could detect the E. coli density with a detection limit of 9.7 × 10² cells/mL within 3 h.     

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.     

Ferric nanoparticle-based resonance light scattering determination of DNA at nanogram levels

A novel assay of DNA has been proposed by using ferric nanoparticles as probes coupled with resonance light scattering (RLS) detection. At pH 7.40, the RLS intensity of ferric nanoparticles can be greatly enhanced by the aggregation of positively charged ferric nanoparticles through electrostatic interaction with negatively charged DNA. The enhanced intensity of RLS at 452 nm is proportional to the concentration of DNA in the range of 0.01-0.8 μg ml⁻¹ for calf thymus and salmon sperm DNA and in the range of 0.005-0.3 μg ml⁻¹ for E. coli K12 genomic DNA. Detection limits are 3.6 ng ml⁻¹ for calf thymus DNA, 4.4 ng ml⁻¹ for salmon sperm DNA, and 1.9 ng ml⁻¹ for E. coli K12 genomic DNA, respectively. Compared with the chromophores previously used in RLS assay, the ferric nanoparticles have offered several advantages in easy preparation, good photostability and high sensitivity without being modified or functionalized.     

Biosensor for chlorogenic acid based on an ionic liquid containing iridium nanoparticles and polyphenol oxidase

A biosensor based on the ionic liquid, 1-n-butyl-3-methylimidazolium hexafluorophosphate containing dispersed iridium nanoparticles (Ir-BMI.PF₆) and polyphenol oxidase was constructed. This enzyme was obtained from the sugar apple (Annona squamosa), immobilized in chitosan ionically crosslinked with oxalate. The biosensor was used for determination of chlorogenic acid by square wave voltammetry. The polyphenol oxidase catalyzes the oxidation of chlorogenic acid to the corresponding o-quinone, which is electrochemically reduced back to this substance at +0.25 V vs. Ag/AgCl. Under optimized operational conditions the chlorogenic acid concentration was linear in the range of 3.48 × 10⁻⁶ to 4.95 × 10⁻⁵ mol L⁻¹ with a detection limit of 9.15 × 10⁻⁷ mol L⁻¹. The biosensor was applied in the determination of chlorogenic acid in organic and decaffeinated coffee and the results compared with those obtained using the capillary electrophoresis method. The recovery study for chlorogenic acid in these samples gave values of 93.2-105.7%.     

Rapid detection of Escherichia coli O157:H7 spiked into food matrices

Food poisoning causes untold discomfort to many people each year. One of the primary culprits in food poisoning is Escherichia coli O157:H7. While most cases cause intestinal discomfort, up to 7% of the incidences lead to a severe complication called hemolytic uremic syndrome which may be fatal. The traditional method for detection of E. coli O157:H7 in cases of food poisoning is to culture the food matrices and/or human stool. Additional performance-based antibody methods are also being used. The NRL array biosensor was developed to detect multiple antigens in multiple samples with little sample pretreatment in under 30 min. An assay for the specific detection of E. coli O157:H7 was developed, optimized and tested with a variety of spiked food matrices in this study. With no sample pre-enrichment, 5 × 10³ cells mL⁻¹ were detected in buffer in less than 30 min. Slight losses of sensitivity (1-5 × 10⁻⁴ cell mL⁻¹⁾ but not specificity occur in the presence of high levels of extraneous bacteria and in various food matrices (ground beef, turkey sausage, carcass wash, and apple juice). No significant difference was observed in the detection of E. coli O157:H7 in typical culture media (Luria Broth and Tryptic Soy Broth).     

DNA impedance biosensor for detection of cancer, TP53 gene mutation,based on gold nanoparticles/aligned carbon nanotubes modified electrode

For the first time, a new platform based on electrochemical growth of Au nanoparticles on aligned multi-walled carbon nanotubes (A-MWCNT) was developed for sensitive lable-free DNA detection of the TP53 gene mutation, one of the most popular genes in cancer research. Electrochemical impedance spectroscopy (EIS) was used to monitor the sequence-specific DNA hybridization events related to TP53 gene. Compared to the bare Ta or MWCNT/Ta electrodes, the synergistic interactions of vertically aligned MWCNT array and gold nanoparticles at modified electrode could improve the density of the probe DNA attachment and resulting the sensitivity of the DNA sensor greatly. Using EIS, over the extended DNA concentration range, the change of charge transfer resistance was found to have a linear relationship in respect to the logarithm of the complementary oligonucleotides sequence concentrations in the wide range of 1.0 × 10⁻¹⁵ − 1.0 × 10⁻⁷ M, with a detection limit of 1.0 × 10⁻¹⁷ M (S/N = 3). The prepared sensor also showed good stability (14 days), reproducibility (RSD = 2.1%) and could be conveniently regenerated via dehybridization in hot water. The significant improvement in sensitivity illustrates that combining gold nanoparticles with the on-site fabricated aligned MWCNT array represents a promising platform for achieving sensitive biosensor for fast mutation screening related to most human cancer types.     

Determination of thiodicarb using a biosensor based on alfalfa sprout peroxidase immobilized in self-assembled monolayers

A biosensor based on alfalfa sprout (Medicago sativa) homogenate as a source of peroxidase is proposed for the determination of thiodicarb by square-wave voltammetry. This enzyme was immobilized in self-assembled monolayers of l-cysteine on a gold electrode. Several parameters were investigated to evaluate the optimum conditions for operation of the biosensor. The analytical curve was linear for thiodicarb concentrations of 2.27 × 10⁻⁶ to 4.40 × 10⁻⁵ mol L⁻¹ with a detection limit of 5.75 × 10⁻⁷ mol L⁻¹. The lifetime of the Au-alfalfa sprout-SAMs was 20 days (at least 220 determinations). The average recovery of thiodicarb from samples of vegetable extracts ranged from 99.02 to 101.04%. The results obtained for thiodicarb in vegetable extracts using the proposed method are in close agreement with those using a high performance liquid chromatography procedure at the 95% confidence level.     

Development of a tyrosinase biosensor based on gold nanoparticles-modified glassy carbon electrodes: Application to the measurement of a bioelectrochemical polyphenols index in wines

The preparation of a tyrosinase biosensor based on the immobilization of the enzyme onto a glassy carbon electrode modified with electrodeposited gold nanoparticles (Tyr-nAu-GCE) is reported. The enzyme immobilized by cross-linking with glutaraldehyde retains a high bioactivity on this electrode material. Under the optimized working variables (a Au electrodeposition potential of −200 mV for 60 s, an enzyme loading of 457 U, a detection potential of −0.10 V and a 0.1 mol l⁻¹ phosphate buffer solution of pH 7.4 as working medium) the biosensor exhibited a rapid response to the changes in the substrate concentration for all the phenolic compounds tested: phenol, catechol, caffeic acid, chlorogenic acid, gallic acid and protocatechualdehyde. A R.S.D. of 3.6% (n = 6) was obtained from the slope values of successive calibration plots for catechol with the same Tyr-nAu-GCE with no need to apply a cleaning procedure to the biosensor. The useful lifetime of one single biosensor was of at least 18 days, and a R.S.D. of 4.8% was obtained for the slope values of catechol calibration plots obtained with five different biosensors. The kinetic constants and the analytical characteristics were calculated for all the phenolic compounds tested. The Tyr-nAu-GCE was applied for the estimation of the phenolic compounds content in red and white wines. A good correlation of the results (r = 0.990) was found when they were plotted versus those obtained by using the spectrophotometric method involving the Folin-Ciocalteau reagent.     

A phenol biosensor based on immobilizing tyrosinase to modified core-shell magnetic nanoparticles supported at a carbon paste electrode

A phenol biosensor was developed based on the immobilization of tyrosinase on the surface of modified magnetic MgFe₂O₄ nanoparticles. The tyrosinase was first covalently immobilized to core-shell (MgFe₂O₄-SiO₂) magnetic nanoparticles, which were modified with amino group on its surface. The resulting magnetic bio-nanoparticles were attached to the surface of carbon paste electrode (CPE) with the help of a permanent magnet. The immobilization matrix provided a good microenvironment for the retaining of the bioactivity of tyrosinase. Phenol was determined by the direct reduction of biocatalytically generated quinone species at −150 mV versus SCE. The resulting phenol biosensor could reach 95% of steady-state current within 20 s and exhibited a high sensitivity of 54.2 μA/mM, which resulted from the high tyrosinase loading of the immobilization matrix. The linear range for phenol determination was from 1 × 10⁻⁶ to 2.5 × 10⁻⁴ M with a detection limit of 6.0 × 10⁻⁷ M obtained at a signal-to-noise ratio of 3. The stability and the application of the biosensor were also evaluated.     

Colorimetric detection of pathogenic bacteria using platinum-coated magnetic nanoparticle clusters and magnetophoretic chromatography

A colorimetric method that uses platinum-coated magnetic nanoparticle clusters (Pt/MNCs) and magnetophoretic chromatography is developed to detect pathogenic bacteria. Half-fragments of monoclonal Escherichia coli O157:H7 (EC) antibodies were functionalized to Pt/MNCs and used to capture E. coli bacteria in milk. After magnetic separation of free Pt/MNCs and Pt/MNC-EC complexes from the milk, a precision pipette was used to imbibe the E. coli-containing solution, then a viscous polyethylene glycol solution. Due to difference in viscosities, the solutions separate into two liquid layers inside the pipette tip. The Pt/MNC-EC complexes were separated from the free Pt/MNCs by applying an external magnetic field, then added to a tetramethylbenzidine (TMB) solution. Catalytic oxidation of TMB by Pt produced color changes of the solution, which enabled identification of the presence of 10 cfu mL⁻¹E. coli bacteria with the naked eye. The total assay time including separation, binding and detection was 30 min.     

A novel tyrosinase biosensor based on hydroxyapatite-chitosan nanocomposite for the detection of phenolic compounds

A novel tyrosinase biosensor based on hydroxyapatite nanoparticles (nano-HA)-chitosan nanocomposite has been developed for the detection of phenolic compounds. The uniform and size controlled nano-HA was synthesized by hydrothermal method, and its morphological characterization was examined by transmission electron microscope (TEM). Tyrosinase was then immobilized on a nano-HA-chitosan nanocomposite-modified gold electrode. Electrochemical impedance spectroscopy and cyclic voltammetry were used to characterize the sensing film. The prepared biosensor was applied to determine phenolic compounds by monitoring the reduction signal of the biocatalytically produced quinone species at −0.2 V (vs. saturated calomel electrode). The effects of the pH, temperature and applied potential on the biosensor performance were investigated, and experimental conditions were optimized. The biosensor exhibited a linear response to catechol over a wide concentration range from 10 nM to 7 μM, with a high sensitivity of 2.11 × 10³ μA mM⁻¹ cm⁻², and a limit of detection down to 5 nM (based on S/N = 3). The apparent Michaelis-Menten constants of the enzyme electrode were estimated to be 3.16, 1.31 and 3.52 μM for catechol, phenol and m-cresol, respectively. Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.     

Platinum nanoparticles functionalized nitrogen doped graphene platform for sensitive electrochemical glucose biosensing

In this work, we reported an efficient platinum nanoparticles functionalized nitrogen doped graphene (PtNPs@NG) nanocomposite for devising novel electrochemical glucose biosensor for the first time. The fabricated PtNPs@NG and biosensor were characterized using transmission electron microscopy, high-resolution transmission electron microscopy, X-ray photoelectron spectroscopy, static water contact angle, UV–vis spectroscopy, electrochemical impedance spectra and cyclic voltammetry, respectively. PtNPs@NG showed large surface area and excellent biocompatibility, and enhanced the direct electron transfer between enzyme molecules and electrode surface. The glucose oxidase (GOx) immobilized on PtNPs@NG nanocomposite retained its bioactivity, and exhibited a surface controlled, quasi-reversible and fast electron transfer process. The constructed glucose biosensor showed wide linear range from 0.005 to 1.1 mM with high sensitivity of 20.31 mA M⁻¹ cm⁻². The detection limit was calculated to be 0.002 mM at signal-to-noise of 3, which showed 20-fold decrease in comparison with single NG-based electrochemical biosensor for glucose. The proposed glucose biosensor also demonstrated excellent selectivity, good reproducibility, acceptable stability, and could be successfully applied in the detection of glucose in serum samples at the applied potential of −0.33 V. This research provided a promising biosensing platform for the development of excellent electrochemical biosensors.     

Lectin sensitized anisotropic silver nanoparticles for detection of some bacteria

A method of bacteria detection by sensitized anisotropic silver nanoparticles is presented. Anisotropic silver nanoparticles with two bands of surface plasmon resonance (SPR) are prepared and sensitized with potato lectin. These nanoparticles are able to detect three bacterial species: Escherichia coli, Bacillus subtilis and Staphylococcus aureus. The interaction of these bacteria with such nanoparticles induces drastic changes in optical spectra of nanoparticles that are correlated with bacteria titer. The maximal sensitivity is observed for S. aureus (up to 1.5 × 10⁴ mL⁻¹).     

A label-free electrochemical biosensor based on a DNA aptamer against codeine

In order to develop a sensor for opium alkaloid codeine detection, DNA aptamers against codeine were generated by SELEX (systematic evolution of ligands by exponential enrichment) technique. An aptamer HL7-14, which is a 37-mer sequence with K_d values of 0.91 μM, was optimized by the truncation-mutation assay. The specificity investigation shows that HL7-14 exhibits high specificity to codeine over morphine, and almost cannot bind to other small molecule. With this new selected aptamer, a novel electrochemical label-free codeine aptamer biosensor based on Au-mesoporous silica nanoparticles (Au-MSN) as immobilized substrate has been proposed using [Fe(CN)₆]^3−/4− as electroactive redox probe. The linear range covered from 10 pM to 100 nM with correlation coefficient of 0.9979 and the detection limit was 3 pM. Our study demonstrates that the biosensor has good specificity, stability and well regeneration. It can be used to detect codeine.     

Functionalized graphene oxide for the fabrication of paraoxon biosensors

There is an increasing need to develop biosensors for the detection of harmful pesticide residues in food and water. Here, we report on a versatile strategy to synthesize functionalized graphene oxide nanomaterials with abundant affinity groups that can capture histidine (His)-tagged acetylcholinesterase (AChE) for the fabrication of paraoxon biosensors. Initially, exfoliated graphene oxide (GO) was functionalized by a diazonium reaction to introduce abundant carboxyl groups. Then, N_α,N_α-bis(carboxymethyl)-l-lysine hydrate (NTA-NH₂) and Ni²⁺ were anchored onto the GO based materials step by step. AChE was immobilized on the functionalized graphene oxide (FGO) through the specific binding between Ni-NTA and His-tag. A low anodic oxidation potential was observed due to an enhanced electrocatalytic activity and a large surface area brought about by the use of FGO. Furthermore, a sensitivity of 2.23 μA mM⁻¹ to the acetylthiocholine chloride (ATChCl) substrate was found for our composite covered electrodes. The electrodes also showed a wide linear response range from 10 μM to 1 mM (R² = 0.996), with an estimated detection limit of 3 μM based on an S/N = 3. The stable chelation between Ni-NTA and His-tagged AChE endowed our electrodes with great short-term and long-term stability. In addition, a linear correlation was found between paraoxon concentration and the inhibition response of the electrodes to paraoxon, with a detection limit of 6.5 × 10⁻¹⁰ M. This versatile strategy provides a platform to fabricate graphene oxide based nanomaterials for biosensor applications.     

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.     

Fluorescence detection of total count of Escherichia coli and Staphylococcus aureus on water-soluble CdSe quantum dots coupled with bacteria

The aim of this paper was to demonstrate a fluorescence measurement method for rapid detection of two bacterial count by using water-soluble quantum dots (QDs) as a fluorescence marker, and spectrofluorometer acted as detection apparatus, while Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were as detection target bacteria. Highly luminescent water-soluble CdSe QDs were first prepared by using thioglycolic acid (TGA) as a ligand, and were then covalently coupled with target bacteria. The bacterial cell images were obtained using fluorescence microscopy. Our results showed that CdSe QDs prepared in water phase were highly luminescent, stable, and successfully conjugated with E. coli and S. aureus. The fluorescence method could detect 10²-10⁷ CFU/mL total count of E. coli and S. aureus in 1-2 h and the low detection limit is 10² CFU/mL. A linear relationship of the fluorescence peak intensity and log total count of E. coli and S. aureus have been established using the equation Y = 118.68X − 141.75 (r = 0.9907).     

Electrochemical deoxyribonucleic acid biosensor based on carboxyl functionalized graphene oxide and poly-l-lysine modified electrode for the detection of tlh gene sequence related to vibrio parahaemolyticus

A carboxyl functionalized graphene oxide (GO-COOH) and electropolymerized ploy-l-lysine (PLLy) modified glassy carbon electrode (GCE) was fabricated and used for the construction of an electrochemical deoxyribonucleic acid (DNA) biosensor. The NH₂ modified probe ssDNA sequences were immobilized on the surface of GO-COOH/PLLy/GCE by covalent linking with the formation of amide bonds, which was stable and furthur hybridized with the target ssDNA sequence. Differential pulse voltammetry (DPV) was used to monitor the hybridization events with methylene blue as electrochemical indicator, which gave a sensitive reduction peak at −0.287 V (vs. SCE). Under the optimal conditions the reduction peak current was proportional to the concentration of tlh gene sequence in the range from 1.0 × 10⁻¹² to 1.0 × 10⁻⁶ mol L⁻¹ with a detection limit as 1.69 × 10⁻¹³ mol L⁻¹ (3σ). The polymerase chain reaction products of tlh gene from oyster samples were detected with satisfactory results, indicating the potential application of this electrochemical DNA sensor.