A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay

A colorimetric sandwich-type assay based on enzyme-linked aptamer assay has been developed for the fast and sensitive detection of as low as 25 fM of thrombin with high linearity. Aptamer-immobilized glass was used to capture the target analyte, whereas a second aptamer, functionalized with horseradish peroxidase (HRP), was employed for the conventional 3,5,3′,5′-tetramethylbenzidine (TMB)-based colorimetric detection. Without the troublesome antibody requirement of the conventional enzyme-linked immunosorbent assay (ELISA), as low as 25 fM of thrombin could be rapidly and reproducibly detected. This assay has superior, or at least equal, recovery and accuracy to that of conventional antibody-based ELISA.     

A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin

A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1–18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.     

Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme-linked aptamer

Here, we describe the fabrication of an electrochemical immunoglobulin E (IgE) aptasensor using enzyme-linked aptamer in the sandwich assay method and thionine as redox probe. In this protocol, 5′-amine-terminated IgE aptamer and thionine were covalently attached on glassy carbon electrode modified with carbon nanotubes/ionic liquid/chitosan nanocomposite. Furthermore, another IgE aptamer was modified with biotin and enzyme horseradish peroxidase (HRP), which attached to the aptamer via biotin–streptavidin interaction. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry were performed at each stage of the chemical modification process to confirm the resulting surface changes. The presence of IgE induces the formation of a double aptamer sandwich structure on the electrode, and the electrocatalytic reduction current of thionine in the presence of hydrogen peroxide was measured as the sensor response. Under optimized conditions and using differential pulse voltammetry as the measuring technique, the proposed aptasensor showed a low detection limit (6 pM) and high sensitivity (1.88 μA nM⁻¹). This aptasensor also exhibited good stability and high selectivity for IgE detection without an interfering effect of some other proteins such as bovine serum albumin (BSA) and lysozyme. The application of the aptasensor for IgE detection in human serum sample was also investigated. The proposed protocol is quite promising as an alternative sandwich approach for various protein assays.     

A conformational switch‐based aptasensor for the chemiluminescence detection of microRNA

A simple microRNA (miRNA) aptasensor has been developed combining the conformational switch of a streptavidin aptamer and isothermal strand displacement amplification. In the presence of its target miRNA, the allosteric molecular beacon (aMB) probe immobilized on the plate can be ‘switched on' and release the streptavidin aptamer. At the same time, Klenow fragment (3′→5′ exo‐) is utilized to initiate DNA‐strand displacement, which starts the target recycling process. Based on the aptamer' high binding affinity and subsequent catalytic chemiluminescence (CL) detection, this CL strategy is highly specific in distinguishing mature miRNAs in same family. It exhibits a dynamic range of four orders of magnitude with a detection limit of 50 fM, and shows great potential for miRNA‐related clinical practices and biochemical research.     

Aptamer-graphene oxide for highly sensitive dual electrochemical detection of Plasmodium lactate dehydrogenase

A 90 mer ssDNA aptamer (P38) enriched against Plasmodium falciparum lactate dehydrogenase (PfLDH) through SELEX process was immobilized over glassy carbon electrode (GCE) using graphene oxide (GO) as an immobilization matrix, and the modified electrode was investigated for detection of PfLDH. The GO was synthesized from powdered pencil graphite and characterized by XRD based on the increased interlayer distance between graphitic layers from 0.345 nm for graphite to 0.829 nm for GO. The immobilization of P38 on GO was confirmed by I_D/I_G intensity ratio in Raman spectra where, the ratio were 0.67, 0.915, and 1.35 for graphite, GO and P38-GO, respectively. The formation of the P38 layer over GO-GCE was evident from an increase in the surface height in AFM analysis of the electrode from ∼3.5 nm for GO-GCE to ∼27 nm for P38-GO-GCE. The developed aptasensor when challenged with the target, a detection of as low as 0.5 fM of PfLDH was demonstrated. The specificity of the aptasensor was confirmed through a voltametric measurement at 0.65 V of the reduced co-factor generated from the PfLDH catalysis. Studies on interference from some common proteins, storage stability, repeatability and analysis of real samples demonstrated the practical application potential of the aptasensor.     

A highly sensitive fluorescence sensor based on lucigenin/chitosan/SiO2 composite nanoparticles for microRNA detection using magnetic separation

In this paper, a convenient reverse‐phase microemulsion method for the synthesis of SiO₂ nanoparticles (NPs) by simply introducing the chitosan and fluorescent dye of lucigenin during the formation reaction of SiO₂ NPs was proposed. Addition of chitosan can make the SiO₂ NPs porous, and increases lucigenin molecule incorporation into chitosan/SiO₂ NPs nanopores based on electrostatic interaction and supermolecular forces. Therefore, fluorescence quantum yield of the lucigenin/chitosan/SiO₂ composite nanoparticles was increased by introduction of chitosan and compared with lucigenin/SiO₂ NPs without chitosan. Because the number of negative charges carried when using single‐stranded DNA (ssDNA) was different from that of double‐stranded DNA (dsDNA), the numbers of lucigenin/chitosan/SiO₂ composite nanoparticles with positive charge adsorbed using ssDNA or dsDNA were different. Consequently, fluorescence intensity caused using ssDNA or dsDNA/miRNA was clearly discriminative. With increase in target DNA/miRNA concentration, the difference in fluorescence intensity also increased, resulting in a good linear relationship between fluorescence intensity sensitizing value and target miRNA concentrations. Therefore, a new fluorescence analysis method for direct detection of let‐7a in human gastric cancer cell samples without enzyme, label free and no immobilization was established using lucigenin/chitosan/SiO₂ composite nanoparticles as a DNA hybrid indicator. The proposed method had high sensitivity and selectivity, low cost and the detection limit was 10 fM (S/N = 3).     

An ultrasensitive supersandwich electrochemical DNA biosensor based on gold nanoparticles decorated reduced graphene oxide

In this article, a supersandwich-type electrochemical biosensor for sequence-specific DNA detection is described. In design, single-strand DNA labeled with methylene blue (MB) was used as signal probe, and auxiliary probe was designed to hybridize with two different regions of signal probe. The biosensor construction contained three steps: (i) capture DNA labeled with thiol was immobilized on the surface of gold nanoparticles decorated reduced graphene oxide (Au NPs/rGO); (ii) the sandwich structure formation contained “capture–target–signal probe”; and (iii) auxiliary probe was introduced to produce long concatamers containing signal molecule MB. Differential pulse voltammetry (DPV) was used to monitor the DNA hybridization event using peak current changes of MB in phosphate-buffered saline (PBS) containing 1.0 M NaClO₄. Under optimal conditions, the peak currents of MB were linear with the logarithm of the concentration of target DNA in the range of 0.1 μM to 0.1 fM with a detection limit of 35 aM (signal/noise = 3). In addition, this biosensor exhibited good selectivity even for single-base mismatched target DNA detection.     

A novel fluorescent aptasensor for the detection of theophylline based on cryonase-driven signal amplification strategy

In the study, we have developed an expedient and efficient method for the detection of theophylline based on the amplification of the signal intensity of fluorescence based on oxidized single-walled carbon nanohorns (oxSWCNHs)/cryonase. When theophylline was not present in the system, oxSWCNHs can adequately adsorb nucleic acid probes labeled by carboxyfluorescein (FAM). In the presence of theophylline, the nucleic acid probe forms the tertiary probe–theophylline complex, which detaches from the surface of the oxSWCNHs. Then, upon reaction with cryonase, the complex can release the FAM and theophylline into the next cycle. The fluorescence signal of the system exhibits a 1:N magnification, enabling quantitative detection of theophylline. The linear range was 30–150 ng/mL, and the limit of detection (LOD) was 6.04 ng/mL. At the same time, it can also be used to detect theophylline in mouse serum.     

Ultra pH‐sensitive detection of total and free prostate‐specific antigen using electrochemical aptasensor based on reduced graphene oxide/gold nanoparticles emphasis on TiO2/carbon quantum dots as a redox probe

The development of a rapid, sensitive, and straightforward detection method of prostate‐specific antigen (PSA) is indispensable for the early diagnosis of prostate cancer (PCa). This work relates an electrochemical method using functionalized single‐stranded DNA aptamer to diagnose PCa and benign prostate hyperplasia. The sensing platform relies on PSA recognition by aptamer/Au/GO‐nanohybrid‐modified glassy carbon electrode. Besides ferrocyanide TiO₂/carbon quantum dots (CQDs) probe is used to investigate the effect of nanoparticle‐containing electrolyte. Optimization of incubation time of aptamer/Au/GO‐nanohybrid and volume fraction of nafion were done using Design Expert 10 software reporting 42.4 h and 0.095% V/V, respectively. In ferrocyanide medium, PSA detection as low as 3, 2.96, and 0.85 ng mL⁻¹ was achieved with a dynamic range from 0.5 to 7 ng ml⁻¹, in accord with clinical values, using cyclic voltammetry, square wave voltammetry, and electrochemical impedance spectroscopy, respectively. Moreover, this sensor exhibited conspicuous performance in TiO₂/CQDs‐containing medium with different pH values of 5.4 and 8 to distinguish total PSA and free PSA, resulting in very low limit of detections, 0.028, and 0.007 ng ml⁻¹, respectively. The results manifested the proposed system as a forthcoming sensor in a clinical and point of care analysis of PSA.     

An electrochemical DNA biosensor based on gold nanorods decorated graphene oxide sheets for sensing platform

A simple electrochemical sensor for sensitive and selective DNA detection was constructed based on gold nanorods (Au NRs) decorated graphene oxide (GO) sheets. The high-quality Au NRs–GO nanocomposite was synthesized via the electrostatic self-assembly technique, which is considered a potential sensing platform. Differential pulse voltammetry was used to monitor the DNA hybridization event using methylene blue as an electrochemical indicator. Under optimal conditions, the peak currents of methylene blue were linear with the logarithm of the concentrations of complementary DNA from 1.0 × 10⁻⁹ to 1.0 × 10⁻¹⁴ M with a detection limit of 3.5 × 10⁻¹⁵ M (signal/noise = 3). Moreover, the prepared electrochemical sensor can effectively distinguish complementary DNA sequences in the presence of a large amount of single-base mismatched DNA (1000:1), indicating that the biosensor has high selectivity.     

A highly sensitive detection of chloramphenicol based on chemiluminescence immunoassays with the cheap functionalized Fe3O4@SiO2 magnetic nanoparticles

A strategy has been applied to chloramphenicol (CAP) detection with chemiluminescence immunoassays (CLIA) based on cheap functionalized Fe₃O₄@SiO₂ magnetic nanoparticles (Fe–MNPs). The strategy that bovine serum albumin (BSA) was immobilized on cheap functionalized Fe–MNPs and that the CAP molecules were then immobilized on BSA, avoided the long process of dialysis for preparation of the BSA‐CAP conjugates. The samples were detected for both methods that utilized two different kinds of functionalized Fe–MNPs (amine‐functionalized Fe₃O₄@SiO₂ and carboxylic acid‐functionalized Fe₃O₄@SiO₂). The sensitivities and limits of detection (LODs) of the two methods were obtained and compared based on inhibition curves. The 50% inhibition concentrations (IC₅₀) values of the two methods were about 0.024 ng ml^?1 and 0.046 ng ml^?1 respectively and LODs were approximately 0.0002 ng ml^?1 and 0.001 ng ml^?1 respectively. These methods were much more sensitive than that of any traditional enzyme‐linked immunosorbent assay (ELISA) previously reported. Therefore, such chemiluminescence methods could be easily adapted for small molecule detection in a variety of foods using Fe–MNPs.     

Colorimetric aptasensor for progesterone detection based on surfactant-induced aggregation of gold nanoparticles

This paper proposes an aptasensor for progesterone (P4) detection in human serum and urine based on the aggregating behavior of gold nanoparticles (AuNPs) controlled by the interactions among P4-binding aptamer, target P4 and cationic surfactant hexadecyltrimethylammonium bromide (CTAB). The aptamer can form an aptamer-P4 complex with P4, leaving CTAB free to aggregate AuNPs in this aptasensor. Thus, the sensing solution will turn from red (520 nm) to blue (650 nm) in the presence of P4 because P4 aptamers are used up firstly owing to the formation of an aptamer-P4 complex, leaving CTAB free to aggregate AuNPs. However, in the absence of P4, CTAB combines with aptamers so that AuNPs still remain dispersed. Therefore, this assay makes it possible to detect P4 not only by absorbance measurement but also through naked eyes. By monitoring the variation of absorbance and color, a CTAB-induced colorimetric assay for P4 detection was established with a detection limit of 0.89 nM. Besides, the absorbance ratio A650/A520 has a linear correlation with the P4 concentration of 0.89–500 nM. Due to the excellent recoveries in serum and urine, this biosensor has great potential with respect to the visual and instrumental detection of P4 in biological fluids.     

A label-free electrochemiluminescence aptasensor for thrombin based on novel assembly strategy of oligonucleotide and luminol functionalized gold nanoparticles

In the work, a label-free electrochemiluminescence (ECL) aptasensor for the sensitive and selective detection of thrombin was constructed based on target-induced direct ECL signal change by virtue of a novel assembly strategy of oligonucleotide and luminol functionalized gold nanoparticles (luminol-AuNPs). It is the first label-free ECL biosensor based on luminol and its analogs functionalized AuNPs. Streptavidin AuNPs coated with biotinylated DNA capture probe 1 (AuNPs-probe 1) were firstly assembled onto an gold electrode through 1,3-propanedithiol. Then luminol-AuNPs co-loaded with thiolated DNA capture probe 2 and thiolated thrombin binding aptamer (TBA) (luminol-AuNPs-probe 2/TBA) were assembled onto AuNPs-probe 1 modified electrode through the hybridization between capture probes 1 and 2. The luminol-AuNPs-probe 2/TBA acted as both molecule recognition probe and sensing interface. An Au/AuNPs/ds-DNA/luminol-AuNPs/TBA multilayer architecture was obtained. In the presence of target thrombin, TBA on the luminol-AuNPs could capture the thrombin onto the electrode surface, which produced a barrier for electro-transfer and influenced the electro-oxidation reaction of luminol, leading to a decrease in ECL intensity. The change of ECL intensity indirectly reflected the concentration of thrombin. Thus, the approach showed a high sensitivity and a wider linearity for the detection of thrombin in the range of 0.005-50nM with a detection limit of 1.7pM. This work reveals that luminol-AuNPs are ideal platform for label-free ECL bioassays.     

A graphene oxide fluorescent sensing platform for sensitive and specific detecting biomarker of radiation-resistant nasopharyngeal carcinoma

Since microRNA-205 (miR-205) is predictive biomarkers for radiation-resistant of nasopharyngeal carcinoma, monitoring of the dynamic variation of miR-205 is of great interest for developing a personalized strategy for the treatment of NPC. Herein, a method for detection of miR-205 was designed based on graphene oxide (GO) and fluorescent probe. The method was successfully used to sensitively and selectively assay miR-205 in aqueous solution, and a low limit of detection of 1.18 nM was obtained in the range 0–300 nM and R² = 0.990. In addition, the designed platform is specific in that it can distinguish the target miRNA from non-target miRNAs, and even the sequences with single base, double base and three base mismatches. Considering the simplicity and superior sensitivity, it has great potential for clinical application in determining biomarker of radiation-resistant nasopharyngeal carcinoma.     

Chemiluminescence immunoassay for the rapid and sensitive detection of antibody against porcine parvovirus by using horseradish peroxidase/detection antibody‐coated gold nanoparticles as nanoprobes

A rapid, simple, facile, sensitive and enzyme‐amplified chemiluminescence immunoassay (CLIA) method to detect antibodies against porcine parvovirus has been developed. Horseradish peroxidase (HRP) and the detection antibody were simultaneously co‐immobilized on the surface of gold nanoparticles using the electrostatic method to form gold nanoparticle‐based nanoprobes. This nanoprobe was employed in a sandwich‐type CLIA, which enables CL signal readout from enzymatic catalysis and results in signal amplification. The presence of porcine parvovirus infection was determined in porcine parvovirus antibodies by measuring the CL intensity caused by the reaction of HRP–luminol with H₂O₂. Under optimal conditions, the obtained calibration plot for the standard positive serum was approximately linear within the dilution range of 1:80 to 1:5120. The limit of detection for the assay was 1:10,240 (S/N = 3), which is much lower than that typically achieved with an enzyme‐linked immunosorbent assay (1:160; S/N = 3). A series of repeatability measurements using 1:320‐fold diluted standard positive serum gave reproducible results with a relative standard deviation of 4.9% (n = 11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor had an efficiency of 90%, a sensitivity of 93.3%, and a specificity of 87.5% relative to the enzyme‐linked immunosorbent assay results. Copyright © 2013 John Wiley & Sons, Ltd.     

Label-free electrochemical immunosensor based on Nile blue A-reduced graphene oxide nanocomposites for carcinoembryonic antigen detection

In this article, a novel, label-free, and inherent electroactive redox immunosensor for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and Nile blue A (NB) hybridized electrochemically reduced graphene oxide (NB–ERGO) is proposed. The composite of NB–graphene oxide (NB–GO) was prepared by π–π stacking interaction. Then, chronoamperometry was adopted to simultaneously reduce HAuCl₄ and nanocomposites of NB–GO for synthesizing AuNPs/NB–ERGO. The immunosensor was fabricated by capturing CEA antibody (anti-CEA) at this nanocomposite modified electrode. The immunosensor determination was based on the fact that, due to the formation of antigen–antibody immunocomplex, the decreased response currents of NB were directly proportional to the concentrations of CEA. Under optimal conditions, the linear range of the proposed immunosensor was estimated to be from 0.001 to 40 ng ml⁻¹ and the detection limit was estimated to be 0.00045 ng ml⁻¹. The proposed immunosensor was used to determine CEA in clinical serum samples with satisfactory results. The proposed method may provide promising potential application in clinical immunoassays with the properties of facile procedure, stability, high sensitivity, and selectivity.     

In situ enzymatic silver enhancement based on functionalized graphene oxide and layer-by-layer assembled gold nanoparticles for ultrasensitive detection of thrombin

A highly specific in situ amplification strategy was designed for ultrasensitive detection of thrombin by combining the layer-by-layer (LBL) assembled amplification with alkaline phosphatase (ALP) and gold nanoparticles (Au) mediated silver deposition. High-density carboxyl functionalized graphene oxide (FGO) was introduced as a nanocarrier for LBL assembling of alkaline phosphatase decorated gold nanoparticles (ALP-Au), which was further adopted to label thrombin aptamer II. After sandwich-type reaction, numerous ALP were captured onto the aptasensor surface and catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP), which in situ generated ascorbic acid (AA), reducing Ag(+) to Ag nanoparticles (AgNPs) for electrochemical readout. Inspiringly, the in situ amplification strategy with ethanolamine as an effective blocking agent showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal, which was favorable to enhance the sensitivity of aptasensor. Our novel dramatic signal amplification strategy, with a detection limit of 2.7fM, showed about 2-3 orders of magnitude improvement in the sensitivity for thrombin detection compared to other universal enzyme-based electrochemical assay.     

A new electrochemical biosensor for DNA detection based on molecular recognition and lead sulfide nanoparticles

In this paper, we constructed a new electrochemical biosensor for DNA detection based on a molecule recognition technique. In this sensing protocol, a novel dual-labeled DNA probe (DLP) in a stem–loop structure was employed, which was designed with dabcyl labeled at the 3′ end as a guest molecule, and with a Pb nanoparticle labeled at the 5′ end as electrochemical tag to indicate hybridization. One α-cyclodextrin-modified electrode (α-CD/MCNT/GCE) was used for capturing the DNA hybridization. Initially, the DLP was in the “closed” state in the absence of the target, which shielded dabcyl from the bulky α-CD/MCNT/GCE conjugate due to a steric effect. After hybridization, the loop sequence (16 bases) formed a rigid duplex with the target, breaking the relatively shorter stem duplex (6 bases). Consequently, dabcyl was forced away from the Pb nanoparticle and became accessible by the electrode. Therefore, the target hybridization event can be sensitively transduced via detecting the electrochemical reduction current signal of Pb. Using this method, as low as 7.1 × 10⁻¹⁰ M DNA target had been detected with excellent differentiation ability for even a single mismatch.     

Electrochemical cytosensor based on gold nanoparticles for the determination of carbohydrate on cell surface

An electrochemical cytosensor was designed based on the specific recognition of mannosyl on a cell surface to concanavalin A (ConA) and the signal amplification of gold nanoparticles (NPs). By sandwiching a cancer cell between a gold electrode modified with ConA and the gold NPs with ConA and 6-ferrocenylhexanethiol (Fc), the electrochemical cytosensor was established. The cell number and the amount of cell surface mannose moieties were quantified by cyclic voltammetry (CV) analysis of the Fc loaded on the surface of the gold NPs. Since a single gold NP could be loaded with hundreds of Fc, a significant amplification for the detection of target cell was obtained. By using K562 leukemic cells (K562 cells) as a model, the electrochemical response was proportional to the cell concentration in the range from 1.0 × 10² to 1.0 × 10⁷ cells mL⁻¹, showing very high sensitivity. The signal amplification could be further used to evaluate the cell surface mannose moieties, and the amount of mannose moieties on a single living K562 cell was detected to correspond to 4.7 × 10⁹ molecules of free mannose. This strategy presents a promising platform in a highly sensitive cytosensor and convenient estimation of cell surface carbohydrate.     

A sensitive immunochromatographic assay using gold nanoparticles for the semiquantitative detection of prostate-specific antigen in serum

In prostate cancer screening, prostate-specific antigen (PSA) has been utilized as a valuable biomarker. There are routinely used procedures based on enzyme-linked immunosorbent assay (ELISA) for PSA detection. The procedures based on ELISA, however, are time consuming, complicated, and costly. We have developed a rapid, very simple, cost effective and sensitive immunochromatographic assay using gold nanoparticles and evaluated its applications for first screening of prostate cancer in serum samples. The sensitive immunochromatographic assay requires only 40 μL of the serum sample. The assay used is rapid and simple, that it totally takes approx 15 min to complete. The method for sensitive immunochromatographic assay has the other advantage of decreasing the antibody concentration that is used for the test line. In this study, we show the advantage to decrease the antibody concentration and the evaluation of our sensitive immunochromatographic assay for the semiquantitative detection of PSA in serum. The results obtained from 163 serum samples using sensitive immunochromatographic assay are compared with the results obtained using the chemiluminescent enzyme immunoassay (CLEIA) and normal immunochromatographic assay. The results obtained in the sensitive immunochromatographic assay correlated well with the values obtained in CLEIA. We concluded that our sensitive immunochromatographic assay is applicable to the first screening test for the diagnosis of prostate cancer. Our developed sensitive immunochromatographic assay is a promising candidate for diagnosis or research use, which may become commercially available in the near future.