Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk

A novel rapid (20 min) fluorescent lateral flow test for chloramphenicol (CAP) detection in milk was developed. The chosen format is a binding-inhibition assay. Water-soluble quantum dots with an emission peak at 625 nm were applied as a label. Milk samples were diluted by 20 % with phosphate buffer to eliminate the matrix effect. The result of the assay could be seen by eye under UV light excitation or registered by a portable power-dependent photometer. The limit of CAP detection by the second approach is 0.2 ng/mL, and the limit of quantitation is 0.3 ng/mL.

Quantum dot-based immunosensor for the detection of prostate-specific antigen using fluorescence microscopy

A sensitive optical method based on quantum dot (QD) technology is demonstrated for the detection of an important cancer marker, total prostate-specific antigen (TPSA) on a disposable carbon substrate surface. Immuno-recognition was carried out on a carbon substrate using a sandwich assay approach, where the primary antibody (Ab)-protein A complex covalently bound to the substrate surface, was allowed to capture TPSA. After the recognition event, the substrate was exposed to the biotinylated secondary Abs. After incubation with the QD streptavidin conjugates, QDs were captured on the substrate surface by the strong biotin-streptavidin affinity. Fluorescence imaging of the substrate surface illuminated the QDs, and provided a very sensitive tool for the detection of TPSA in undiluted human serum samples with a detection limit of 0.25 ng/mL. The potential of this method for application as a simple and efficient diagnostic strategy for immunoassays is discussed.     

Quantum dot-based isothermal chain elongation for fluorescence detection of specific DNA sequences via template-dependent surface-hybridization

A new quantum dot-based method to detect specific sequences of DNA is proposed. The capture and reporter probes do not hybridize to each other, but in the presence of a template they can anneal to each other via the formation of a stable ternary complex. Because of the specific design of the capture and reporter probes, the 5' end of the template target DNA remains free to hybridize with another reporter. In this way, each capture DNA is an initiator strand that triggers a cascade of hybridization events between the target DNA and the reporter probe. This forms a superstructure, enhances base stacking, and produces a strong fluorescent signal. The introduction of T4 DNA ligase further stabilizes the superstructure and greatly increases the fluorescence intensity, and the detection limit is as low as 10 fM. This fluorescence method is advantageous over conventional techniques because of its excellent ability to discriminate single base-pair mismatches and single nucleotide gap or flap. This simple technique is promising for improving medical diagnosis and treatment.     

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol

An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH = 9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H₂O₂ product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL⁻¹ to 1000 ng mL⁻¹, and a low detection limit was 0.02 ng mL⁻¹. The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.     

Quantum dot-based "turn-on" fluorescent probe for detection of zinc and cadmium ions in aqueous media

Health or environmental issue caused by abnormal level of metal ions like Zn²⁺ or Cd²⁺ is a worldwide concern. Developing an inexpensive and facile detection method for Zn²⁺ and Cd²⁺ is in urgent demand. Due to their super optical properties, fluorescent quantum dots (QDs) have been developed as a promising alternative for organic dyes in fluorescence analysis. In this study, a CdTe QDs-based sensitive and selective probe for Zn²⁺ and Cd²⁺ in aqueous media was reported. The proposed probe worked in fluorescence “turn-on” mode. The initial bright fluorescence of CdTe QDs was effectively quenched by sulfur anions (S²⁻). The presence of Zn²⁺ (or Cd²⁺) can “turn-on” the weak fluorescence of QDs quenched by S²⁻ due to the formation of ZnS (or CdS) passivation shell. Under optimal conditions, a good linear relationship between the fluorescence response and concentration of Zn²⁺ (or Cd²⁺) could be obtained in the range from 1.6 to 35 μM (1.3–25 μM for Cd²⁺). The limit of detection (LOD) for Zn²⁺ and Cd²⁺ were found to be 1.2 and 0.5 μM, respectively. Furthermore, the present probe exhibited a high selectivity for Zn²⁺ and Cd²⁺ over other metal ions and was successfully used in the detection of Zn²⁺ or Cd²⁺ in real water samples.     

Universal quantum dot-based sandwich-like immunoassay strategy for rapid and ultrasensitive detection of small molecules using portable and reusable optofluidic nano-biosensing platform

A universal sandwich-like immunoassay strategy based on quantum-dots immunoprobe (QD-labeled anti-mouse IgG antibody) was developed for rapid and ultrasensitive detection of small molecules. A portable and reusable optofluidic nano-biosensing platform was applied to investigate the sandwich-like immunoassay mechanism and format of small molecules, as well as the binding kinetics between QD immunoprobe and anti-small molecule antibody. A two-step immunoassay method that involves pre-incubation mixture of different concentration of small molecule and anti-small molecule antibody, and subsequent introduction of QD immunoprobe into the optofluidic cell was conducted for small molecule determination. Compared with the one-step immunoassay method, the two-step immunoassay method can obtain higher fluorescence signal and higher sensitivity index, thus improving the nano-biosensing performance. Based on the proposed strategy, two mode targets, namely, microcystin-LR (MC-LR) and Bisphenol A (BPA) were tested with high sensitivity, rapidity, and ease of use. A higher concentration of small molecules in the sample led to less anti-small molecule antibody bound with antigen-carrier protein conjugate immobilized onto the sensor surface, and less QD immunoprobes bound with anti-small molecule antibody. This phenomenon lowered the fluorescence signal detected by nano-biosensing platform. Under optimal operating conditions, MC-LR and BPA exhibited a limit of detection of 0.003 and 0.04 μg/L, respectively. The LODs were better than those of the indirect competitive immunoassay method for small molecules via Cy5.5-labeled anti-small molecule antibody. The proposed QD-based sandwich-like immunoassay strategy was evaluated in spiked water samples, and showed good recovery, precision and accuracy without complicated sample pretreatments. All these results demonstrate that the new detection strategy could be readily applied to the other trace small molecules in real water samples.     

Quantum dot-based energy transfer: perspectives and potential for applications in photodynamic therapy

Quantum dots have emerged as an important class of material that offers great promise to a diverse range of applications ranging from energy conversion to biomedicine. Here, we review the potential of using quantum dots and quantum dot conjugates as sensitizers for photodynamic therapy (PDT). The photophysics of singlet oxygen generation in relation to quantum dot-based energy transfer is discussed and the possibility of using quantum dots as photosensitizer in PDT is assessed, including their current limitations to applications in biological systems. The biggest advantage of quantum dots over molecular photosensitizers that comes into perspective is their tunable optical properties and surface chemistries. Recent developments in the preparation and photophysical characterization of quantum dot energy transfer processes are also presented in this review, to provide insights on the future direction of quantum dot-based photosensitization studies from the viewpoint of our ongoing research.     

Quantum dot-based array for sensitive detection of <Emphasis Type="Italic">Escherichia coli</Emphasis>

A fluorescent quantum dot-based antibody array, used in sandwich format, has been developed to detect Escherichia coli O157:H7. Numerous parameters such as solid support, optimal concentration of immunoreagents, blocking reagents, and assay time were optimized for array construction. Quantum dot-conjugated anti-IgG was used as the detecting system. The array allows the detection of E. coli O157:H7 at concentrations below 10 CFU mL⁻¹ without sample enrichment, exhibiting an increase of three orders of magnitude in the limit of detection compared to ELISA. The interference caused by Gram (+) and Gram (−) bacteria was negligible at low concentrations of bacteria.     

Functionalized gold nanoparticles for ultrasensitive DNA detection

A major challenge in the area of DNA detection is the development of rapid methods that do not require polymerase chain reaction (PCR) amplification of the genetic sample. The PCR amplification step increases the cost of the assay, the complexity of the detection, and the quantity of DNA required for the assay. In this context, methods that are able to perform DNA analyses with ultrasensitivity have recently been investigated with the aim of developing new PCR-free detection protocols. Functionalized gold nanoparticles have played a central role in the development of such methods. Here, possibilities offered by functionalized gold nanoparticle in the ultrasensitive detection of DNA are discussed. The different functionalization protocols available for gold nanoparticles and the principal DNA detection methods that are able to detect DNA at the femtomolar to attomolar level are presented.     

Disposable integrated bismuth citrate-modified screen-printed immunosensor for ultrasensitive quantum dot-based electrochemical assay of C-reactive protein in human serum

A novel immunosensor based on graphite screen-printed electrodes (SPEs) modified with bismuth citrate was developed for the voltammetric determination of C-reactive protein (CRP) in human serum using quantum dots (QDs) labels. The sandwich-type immunoassay involved physisorption of CRP capture antibody on the surface of the sensor, sequential immunoreactions with CRP and biotinylated CRP reporter antibody and finally reaction with streptavidin-conjugated PbS QDs. The quantification of the target protein was performed with acidic dissolution of the PbS QDs and anodic stripping voltammetric detection of the Pb(II) released. Detection was performed at bismuth nanodomains formed on the sensor surface during the electrolytic preconcentration step, as bismuth citrate was reduced to metallic bismuth simultaneously with the deposition of Pb on the surface of the immunosensor. Under optimal conditions, the response was linear over the range 0.2–100 ng mL⁻¹ CRP and the limit of detection was 0.05 ng mL⁻¹ CRP. Since the modified SPE serves as both the biorecognition element and the QDs reader, the analytical procedure is simplified, the drawbacks of existing electroplated immunosensors are minimized while the proposed disposable sensing platform provides convenient, low-cost and ultrasensitive detection of proteins and wider scope for mass-production.     

Functionalized semiconductor nanocrystals for ultrasensitive detection of peptides

Semiconductor CdS nanoparticle have been prepared and modified with thiovanic acid. The functionalized nanoparticles are water-soluble. They were used as the fluorescence probes in the ultrasensitive detection of peptides. This method is based on the fluorescence enhancement of functionalized nano-CdS in the presence of peptide with mercapto groups (GN-9) and the fluorescence quenching of functionalized nano-CdS in the presence of peptide (GA-8 and MT-25). Excitation and emission wavelengths were 360 and 530 nm, respectively. Under optimum conditions, the calibration graphs are linear over the range of 0.15-3.5, 0.2-4.0, and 0.2-3.8 μg ml⁻¹ for GN-9, GA-8 and MT-25, respectively. The corresponding detection limits were 0.010 μg ml⁻¹ for GN-9, 0.018 μg ml⁻¹ for GA-8 and 0.022 μg ml⁻¹ for MT-25, respectively. This method has been proved to be a simple, rapid and sensitive method.     

Aptamer-based polymerase chain reaction for ultrasensitive cell detection

A new system was developed for sensitive and selective detection of tumor cells taking advantage of cell-attached aptamers amplified by PCR and output signals amplified by cationic conjugated polymers.     

Immuno-PCR: An ultrasensitive immunoassay for biomolecular detection

Techniques that combine nucleic acid amplification with an antibody-based assay can dramatically increase the sensitivity of conventional immunoassays. This review summarizes the methodology and applications of one such protein detection technique that has been used for the past 23 years—the immuno-polymerase chain reaction (usually referred to as immuno-PCR or IPCR). The key component of an immuno-PCR is a DNA–antibody conjugate that serves as a bridge to link the solid-phase immunoreaction with nucleic acid amplification. The efficiency of immuno-PCR enables a 10- to 10⁹-fold increase in detection sensitivity compared with that of ELISA. Advancements in immuno-PCR have included improvements of production of the DNA–antibody conjugate, assay formats, and readout methods. As an ultrasensitive protein assay, immuno-PCR has a broad range of applications in immunological research and clinical diagnostics.     

A nanocatalyst-based assay for proteins: DNA-free ultrasensitive electrochemical detection using catalytic reduction of p-nitrophenol by gold-nanoparticle labels

This communication reports a nanocatalyst-based electrochemical assay for proteins. Ultrasensitive detection has been achieved by signal amplification combined with noise reduction: the signal is amplified both by the catalytic reduction of p-nitrophenol to p-aminophenol by gold-nanocatalyst labels and by the chemical reduction of p-quinone imine to p-aminophenol by NaBH4; the noise is reduced by employing an indium tin oxide electrode modified with a ferrocenyl-tethered dendrimer and a hydrophilic immunosensing layer.     

An electronic channel switching-based aptasensor for ultrasensitive protein detection

Due to the ubiquity and essential of the proteins in all living organisms, the identification and quantification of disease-specific proteins are particularly important. Because the conformational change of aptamer upon its target or probe/target/probe sandwich often is the primary prerequisite for the design of an electrochemical aptameric assay system, it is extremely difficult to construct the electrochemical aptasensor for protein assay because the corresponding aptamers cannot often meet the requirement. To circumvent the obstacles mentioned, an electronic channel switching-based (ECS) aptasensor for ultrasensitive protein detection is developed. The essential achievement made is that an innovative sensing concept is proposed: the hairpin structure of aptamer is designed to pull electroactive species toward electrode surface and makes the surface-immobilized IgE serve as a barrier that separates enzyme from its substrate. It seemingly ensures that the ECS aptasensor exhibits most excellent assay features, such as, a detection limit of 4.44 × 10⁻⁶ μg mL⁻¹ (22.7 fM, 220 zmol in 10-μL sample) (demonstrating a 5 orders of magnitude improvement in detection sensitivity compared with classical electronic aptasensors) and dynamic response range from 4.44 × 10⁻⁶ to 4.44 × 10⁻¹ μg mL⁻¹. We believe that the described sensing concept here might open a new avenue for the detection of proteins and other biomacromolecules.     

Spreader-CDR system for efficient,reproducible, and frugal western blot

Efficient antibody incubation is a vital step for successful western blot. During the incubation, a thin antibody-depleted layer is created around the blotting membrane, which limits antibody binding. Although the conventional batch shaking method is ineffective against it, this layer can be easily disrupted by cyclic draining and replenishing (CDR) of the antibody solution during membrane incubation. Previously, we introduced a closed and rotating cylindrical chamber as a tool to implement CDR for western blots (rCDR). A new open bucket-style chamber was devised for easier operation and the possibility of process automation. Instead of rotation as in rCDR, rocking it back and forth achieved the CDR antibody incubation (R-CDR). The chamber was then equipped with a spreader-rod to facilitate the uniform movement of the antibody solution across the membrane surface. Hence, it was named spreader CDR (S-CDR). Compared to the batch incubation method, both the S-CDR and R-CDR devices produced significantly enhanced signals and developed faster results. There were several additional benefits of using the spreader-rod, which included uniform antibody binding across the membrane, reduced usage of antibodies, and the ability to recover results even from mishandled, creased membranes. The S-CDR device ensures better blots and can be easily implemented in existing western blot protocols.     

Fluorescence for the ultrasensitive detection of peptides with functionalized nano-ZnS

The fluorescence emitted by the functionalized ZnS nanocrystal at 440 nm could be efficiently enhanced or quenched when various peptides were added. The mechanism of the fluorescence enchancement and quenching of ZnS nanocrystals was discussed. The binding constant and numbers of binding sites was obtained from the Scatchard plot. The change of fluorescence intensity was in proportion to the concentration of peptides. The limits of detection were in range of 0.011-0.028 μg mL⁻¹. Application results to synthetic samples showed simplicity, rapidity, high sensitivity and satisfactory reproducibility of the presented method. Measurements of real samples also give satisfactory results which were in good agreement with those obtained using high performance liquid chromatograph (HPLC) and liquid chromatograph-mass spectrograph (LC-MS) methods.