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Recent advances in volume electron microscopy (vEM) allow unprecedented visualization of the electron-dense structures of cells, tissues and model organisms at nanometric resolution in three dimensions (3D). Light-based microscopy has been widely used for specific localization of proteins; however, it is restricted by the diffraction limit of light, and lacks the ability to identify underlying structures. Here, we describe a protocol for ultrastructural detection, in three dimensions, of a protein (Connexin 43) expressed in the intercalated disc region of adult murine heart. Our protocol does not rest on the expression of genetically encoded proteins and it overcomes hurdles related to pre-embedding and immunolabeling, such as the penetration of the label and the preservation of the tissue. The pre-embedding volumetric immuno-electron microscopy (pre-embedding vIEM) protocol presented here combines several practical strategies to balance sample fixation with antigen and ultrastructural preservation, and penetration of labeling with blocking of non-specific antigen binding sites. The small 1.4 nm gold along with surrounded silver used as a detection marker buried in the sample also serves as a functional conductive resin that significantly reduces the charging of samples. Our protocol also presents strategies for facilitating the successful cutting of the samples during serial block-face scanning electron microscopy (SBF-SEM) imaging. Our results suggest that the small gold-based pre-embedding vIEM is an ideal labeling method for molecular localization throughout the depth of the sample at subcellular compartments and membrane microdomains.

Staphylococcus aureus is the Gram-positive, immobile, round-shaped bacterium usually existing on wounds, marine environments, and contaminated food. α-Hemolysin (also called -toxin), a common pore-forming toxin that presents in most S. aureus strains with the ability to penetrate the host cell membrane, leading to osmotic swelling and cell death, appears to be a potential biomarker for detection. Based on a sandwich format, this study developed a lateral flow immunoassay (LFIA) targeting -toxin to detect S. aureus. Silver enhancement method was applied to enhance the test line's signal and, therefore, improve the limit of detection (LOD). This method relies on the accumulation on AuNP surface of silver atoms which come from silver nitrate salt after being reduced by reducing agent. The developed LFIA could detect the target antigen within approximately 20 min with no cross-reactivity. The LOD was increased 10-fold after the silver enhancement, from 105 to 104 CFU/mL of bacteria suspensions. The results indicated that -toxin is a promising target to detect S. aureus. Targeting -toxin not only can be a potential detection approach but also a tool for measuring toxicity. [ABSTRACT FROM AUTHOR]

Lead ions are a toxic metal that can enter the human body through the diet and negatively impact human health. A wide variety of foods, such as vegetables, meat, aquatic product, and drinking water, can be contaminated with lead ions. In this study, a highly sensitive immunochromatographic assay (ICA) based on antibody-oriented probe and silver enhancement was established for the qualitative and quantitative determination of lead ions in drinking water. The recombinant protein A (PrA) was used in probe preparation to improve antibody bioactivity while silver enhancement method was applied to enhance the signal reporter of ICA. It was determined that the visual limit of detection and the scanning limit of quantification of the ICA based on antibody-oriented probe and silver enhancement for lead ions in drinking water were 0.2 ng/mL and 0.08 ng/mL, respectively. The results obtained from developed ICA strip and ICP-MS had no obvious difference using statistical analysis of t test. Compared to previously reported ICA methods for lead ions, the developed ICA method enabled analyzing lead ions in drinking water with a superior sensitivity and detection range. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a sensitive lateral flow immunoassay (LFIA) for the rapid detection of Escherichia coli (E. coli) O157:H7, a pathogen contributor to diseases and fatalities worldwide. Au nanoparticles with high stability, uniform size, and shape were synthesized and coated with heterobifunctional PEG polymer with carboxyl groups, and they were bioconjugated to be used as label in sandwich-LFIA. Then, a silver enhancement strategy was developed as an accessible, rapid, and cost-effective approach for signal amplification to reduce the limit of detection (LOD). The optimal results were achieved when a solution of silver nitrate and hydroquinone/citrate buffer was added to the strips for 4 min. This led to a decrease in the visual LOD from 2 × 106 (CFU mL−1) to 2 × 103 (CFU mL−1), resulting in a threefold improvement in sensitivity compared to the conventional LFIA system. The specificity of the system was evaluated by using non-target bacteria (E. coli BL21 and E. coli T515) and its reliability was determined by testing commercial food samples (milk, tap water, and orange juice), demonstrating its effectiveness for quickly detecting pathogenic bacteria in food products. [ABSTRACT FROM AUTHOR]

Acetaminophen (N-acetyl-p-aminophenol, APAP) is regularly used for antipyretic and analgesic purposes. Overdose or long-term exposure to APAP could lead to liver damage and hepatotoxicity. In this study, the approach of enhanced electrochemical detection of APAP by nanostructured biomass carbon/silver was developed. Porous biomass carbon derived from Elaeagnus Angustifolia gum was prepared by pyrolysis with co-doping of electron-rich elements of nitrogen, sulfur, and phosphorus. The electrodeposition of silver onto a glassy carbon electrode modified with porous carbon could enhance the sensing signal towards APAP. Two linear ranges from 61 nM to 500 μM were achieved with a limit of detection of 33 nM. The developed GCE sensor has good anti-interference, stability, reproducibility, and human urine sample analysis performance. The silver-enhanced biomass carbon GCE sensor extends the application of biomass carbon, and its facile preparation approach could be used in constructing disposable sensing chips in the future. [ABSTRACT FROM AUTHOR]

A novel colorimetric aptasensor based on charge effect-assisted silver enhancement was developed to detect ochratoxin A (OTA). To achieve this objective, gold nanoparticles (AuNPs), which can catalyze silver reduction and deposition, were used as the carrier of the aptamers tagged with a positively charged tetramethylrhodamine (TAMRA). Due to the mutual attraction of positive and negative charges, the TAMRA attracted and retained the silver lactate around the AuNPs. Thus, the chance of AuNP-catalyzed silver reduction was increased. The charge effect-assisted silver enhancement was verified by tagging different base pair length aptamers with TAMRA. Under optimized conditions, the as-prepared OTA aptasensor had a working range of 1 × 102–1 × 106 pg mL−1. The detection limit was as low as 28.18 pg mL−1. Moreover, the proposed aptasensor has been successfully applied to determine OTA in actual samples with satisfactory results. [ABSTRACT FROM AUTHOR]

Acetaminophen (N-acetyl-p-aminophenol, APAP) is regularly used for antipyretic and analgesic purposes. Overdose or long-term exposure to APAP could lead to liver damage and hepatotoxicity. In this study, the approach of enhanced electrochemical detection of APAP by nanostructured biomass carbon/silver was developed. Porous biomass carbon derived from Elaeagnus Angustifolia gum was prepared by pyrolysis with co-doping of electron-rich elements of nitrogen, sulfur, and phosphorus. The electrodeposition of silver onto a glassy carbon electrode modified with porous carbon could enhance the sensing signal towards APAP. Two linear ranges from 61 nM to 500 μM were achieved with a limit of detection of 33 nM. The developed GCE sensor has good anti-interference, stability, reproducibility, and human urine sample analysis performance. The silver-enhanced biomass carbon GCE sensor extends the application of biomass carbon, and its facile preparation approach could be used in constructing disposable sensing chips in the future.

A novel colorimetric aptasensor based on charge effect-assisted silver enhancement was developed to detect ochratoxin A (OTA). To achieve this objective, gold nanoparticles (AuNPs), which can catalyze silver reduction and deposition, were used as the carrier of the aptamers tagged with a positively charged tetramethylrhodamine (TAMRA). Due to the mutual attraction of positive and negative charges, the TAMRA attracted and retained the silver lactate around the AuNPs. Thus, the chance of AuNP-catalyzed silver reduction was increased. The charge effect-assisted silver enhancement was verified by tagging different base pair length aptamers with TAMRA. Under optimized conditions, the as-prepared OTA aptasensor had a working range of 1 × 102–1 × 106 pg mL−1. The detection limit was as low as 28.18 pg mL−1. Moreover, the proposed aptasensor has been successfully applied to determine OTA in actual samples with satisfactory results.

Background: Staphylococcus aureus is one of the most important microorganisms that can colonize the human body and cause various types of human diseases by secreting virulence factors. The lateral flow assay (LFA) is one of the well-known commercial immunoassay methods that provides a high sensitive and rapid approach to monitoring infectious agents in blood, serum and urine. LFA has been considered as an ideal immunochromatographic test. Materials & Methods: Anti Staphylococcal Enterotoxin B (SEB) antibodies were conjugated to 20 nm colloidal gold nanoparticles and were applied in assembled lateral flow layer. Suitable reagents were prepared and used in silver enhancement method. We designed a single immunochromatographic test strip to detect SEB. Results: In this study, the smallest amount of SEB identified using sandwich LFA method was 10 ng/mL. We also established a "silver enhanced method". Silver could improve the sensitivity detection of the test 100-fold greater than the previously mentioned sandwich LFA. Conclusion: Regarding the high sensitivity of the new method for detection and measurement of SEB (0.1 ng/mL), this strip test offers great promise for a rapid technique instead of the other diagnostic SEB tests in laboratories for the first time.

Glycocalyx (GCX) is a thin layer of negatively charged glycoproteins that covers the vascular endothelial surface and regulates various biological processes. Because of the delicate and fragile properties of this structure, it is difficult to detect GCX morphologically. We established a simple method for a three-dimensional visualization of endothelial GCX using low-vacuum scanning electron microscopy (LVSEM) on formalin-fixed paraffin-embedded (FFPE) sections. Mouse kidney tissue was fixed with 10% buffered formalin containing 1% Alcian blue (ALB) via perfusion and immersion. FFPE sections were observed by light microscopy (LM) and LVSEM, and formalin-fixed epoxy resin-embedded ultrathin sections were observed by transmission electron microscopy (TEM). The endothelial GCX from various levels of kidney blood vessels was stained blue in LM and confirmed as a thin osmiophilic layer in TEM. In LVSEM, the sections stained by periodic acid methenamine silver (PAM) revealed the endothelial GCX as a layer of dense silver-enhanced particles, in both the samples fixed via perfusion and immersion. Correlative light and electron microscopy (CLEM) revealed the fine visible structure of endothelial GCX. This simple method using FFPE samples with ALB will enable the three-dimensional evaluation of endothelial GCX alterations in various human diseases associated with endothelial injury in future studies. [ABSTRACT FROM AUTHOR]

Rapid non-laboratory screening of plants for pathogenic viruses crucially influences crop yields in modern agricultural technologies. The aim of this study was to develop a highly-sensitive lateral flow immunoassay (LFIA) for rapid detection of potato leafroll virus (PLRV), an infectious agent of one of the most widespread potato diseases. The proposed LFIA combines the formation of sandwich immune complexes with gold nanoparticles (GNP) as labels and silver enhancement. The enhancement stage was realized using mixture of silver lactate and hydroquinone and subsequent addition of chloride-containing buffer to stop silver reduction. LFIA with silver enhancement was 15 times more sensitive (detection limit 0.2 ng/mL; 15 min) compared with conventional LFIA (detection limit 3 ng/mL; 10 min). The enhanced LFIA detected PLRV in leaves’ extracts of infected potato in dilutions higher than enzyme-linked immunosorbent assay.

Double staining protocols using the most popular immunoperoxidase techniques may raise difficulties. The two ordinary detection systems may cross-talk, when the primary antibodies are derived from phylogenetically closely related animals. A color shift of the 3,3′-diaminobenzidine (DAB) polymer may occur during the second development, resulting in poor distinction between the two kinds of deposits. A post-DAB technique, sulfide-silver-gold intensification, was fine tuned to eliminate these difficulties, which may be especially suitable for colocalization of cell nuclei and perikarya of the same cells. The revised method was probed in combination with a subsequent other immunoperoxidase step or fluorochrome-tagged reagents. The nuclear antigens (BrdU, c-Fos, and Prox-1) were first visualized with DAB polymer, which were then treated with SSGI, turning the deposit black. Thereafter, cytoplasmic antigens (doublecortin, neuronal nuclei, and calbindin) were detected with either another immunoperoxidase using DAB again or immunofluorescence labeling. In both approaches, the immunopositive nuclei and cytoplasmic sites could be easily distinguished even at low magnifications. Different shielding or eluting posttreatments were compared for consecutive acetylcholinesterase histochemistry terminated with DAB development and immunohistochemistry in the same sections. In conclusion, we recommend post-DAB treatments that abolish interactions between detection systems and allow clear distinction between the two signals under various conditions: [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus is one of the most important microorganisms that can colonize the human body and cause various types of human diseases by secreting virulence factors. The lateral flow assay (LFA) is one of the well-known commercial immunoassay methods that provides a high sensitive and rapid approach to monitoring infectious agents in blood, serum and urine. LFA has been considered as an ideal immunochromatographic test. Methods: Anti Staphylococcal Enterotoxin B (SEB) antibodies were conjugated to 20 nm colloidal gold nanoparticles and were applied in assembled lateral flow layer. Suitable reagents were prepared and used in silver enhancement method. We designed a single immunochromatographic test strip to detect SEB. Results: In this study, the smallest amount of SEB identified using sandwich LFA method was 10 ng/mL. We also established a "silver enhanced method". Silver could improve the sensitivity detection of the test 100-fold greater than the previously mentioned sandwich LFA. Conclusion: Regarding the high sensitivity of the new method for detection and measurement of SEB (0.1 ng/mL), this strip test offers great promise for a rapid technique instead of the other diagnostic SEB tests in laboratories for the first time. [ABSTRACT FROM AUTHOR]

Abstract Lateral flow immunoassay (LFIA) is a widely used express method and offers advantages such as a short analysis time, simplicity of testing and result evaluation. However, an LFIA based on gold nanospheres lacks the desired sensitivity, thereby limiting its wide applications. In this study, spherical nanogold labels along with new types of nanogold labels such as gold nanopopcorns and nanostars were prepared, characterized, and applied for LFIA of model protein antigen procalcitonin. It was found that the label with a structure close to spherical provided more uniform distribution of specific antibodies on its surface, indicative of its suitability for this type of analysis. LFIA using gold nanopopcorns as a label allowed procalcitonin detection over a linear range of 0.5–10 ng mL−1 with the limit of detection of 0.1 ng mL−1, which was fivefold higher than the sensitivity of the assay with gold nanospheres. Another approach to improve the sensitivity of the assay included the silver enhancement method, which was used to compare the amplification of LFIA for procalcitonin detection. The sensitivity of procalcitonin determination by this method was 10 times better the sensitivity of the conventional LFIA with gold nanosphere as a label. The proposed approach of LFIA based on gold nanopopcorns improved the detection sensitivity without additional steps and prevented the increased consumption of specific reagents (antibodies).

Analytical method for simultaneous detection of ofloxacin and lomefloxacin in milk by visualized microarray immunoassay without the need of time-consuming or complex pre-treatment steps was reported. A 96-well microplate was used as solid support, in which ofloxacin antigen and lomefloxacin antigen were immobilized, respectively. After immobilization, a mixture of standard solutions containing the analytes or samples and relevant antibodies were added to the array reaction areas, then added silver nanoparticles (AgNPs) labeled secondary antibody (goat anti-mouse IgG). Silver enhancement technique was applied to amplify the detection signals, which produced black image on array spots visible with naked eyes. The signals were detected with a microarray scanner; therefore the analyte residues could detect quantitatively by standard calibration curve. Ofloxacin and lomefloxacin dynamic range were all from 0.05 ng/mL to 12.8 ng/mL. The calibration curve of ofloxacin was calculated as: y = − 0.3446x + 0.4287, r = 0.9849. The calibration curve of lomefloxacin was calculated as: y = − 0.3055x + 0.4797, r = 0.9927. The spike recovery were within 80–130%, and RSD were less than 15%. The limits of detection (LOD) were estimated to be 0.24 ng/mL (ofloxacin) and 0.35 ng/mL (lomefloxacin) (n = 3, 3SD). [ABSTRACT FROM AUTHOR]

[Display omitted] • An ultrasensitive electrochemical DNA biosensor coupled with dual amplification strategy was developed. • DNA-Au bio-bar codes improved the hybridization efficiency and reduced the cross reaction between the targets and reporter DNA. • AgNPs deposited on AuNPs exhibited a dramatic enhancement of DNA hybridization signal. • The DNA biosensor achieved sensitive detection of MDR1 gene with a detection limit of 33 fM (S/N = 3). Given the tumors might develop multidrug resistance (MDR) during chemotherapy that leading to treatment failure, rapid and sensitive determination of MDR1 gene is an effective tool to monitor the development of MDR. Herein, we established an electrochemical deoxyribonucleic acid (DNA) biosensor coupled with dual amplification strategy based on DNA-Au bio-bar codes and enhancement of silver for sensitive and selective determination of MDR1 gene. Specifically, DNA-Au bio-bar codes as signal probe were prepared by immobilizing complementary and non-complementary sequences on gold nanoparticles (AuNPs), so as to reduce the cross reaction between the target and reporter DNA. In addition, AuNPs-catalyzed reduction of silver ions greatly amplified the current signal. Under the optimal conditions, the current signal was proportional to the base-10 logarithm of target DNA concentration in a wide range from 100 fM to 1 nM with a detection limit as low as 33 fM (S/N = 3). Moreover, without the need for target amplification, the prepared biosensor exhibited superior selectivity and high sensitivity even in the presence of other sequences. Therefore, the developed electrochemical DNA biosensor is a facile and sensitive platform for MDR1 gene detection. [ABSTRACT FROM AUTHOR]

Highlights • Development, comparison and validation of silver enhanced, multiplex sandwich vertical flow immunogold assay (SVIA) for sensitive and rapid detection of OTC residues in edible fish tissues. • Combining silver enhancement with immunogold labelled MAb improved the sensitivity. • Employing anti-OTC capture rabbit antiserum and detector monoclonal antibody provided high specificity with a visual detection limit of 2 ng mL−1. • It is a semi-quantitative, simple, rapid, low cost assay compatible with field level analysis. Abstract A rapid multiplex silver enhanced ‘sandwich vertical flow immunogold assay (SVIA)’ based on disposable porous filter-membrane was developed for on-site detection of oxytetracycline (OTC) residues in fish tissues. Artificial antigens were synthesized to raise selective anti-OTC rabbit antiserum and highly specific monoclonal antibody (mAb). The assay consists of three layers. The first layer was composed of immobilized anti-OTC rabbit antiserum (capture antibody) onto the membrane, the second layer was the molecule of interest (OTC) and the third layer was refined with detector immunogold labeled mAb. The sensitivity of the silver enhanced SVIA was as low as 2 ng mL−1 (within 4 min) representing a 125-fold increase in sensitivity over the HRP labeled sandwich vertical flow assay. The reliability of developed technique was co-evaluated with HPLC and validated using 115 field fish samples from different regions of southern India. The results demonstrated that SVIA has the high potentiality to be established as a semi-quantitative routine on-site screening tool. [ABSTRACT FROM AUTHOR]

Rapid non-laboratory screening of plants for pathogenic viruses crucially influences crop yields in modern agricultural technologies. The aim of this study was to develop a highly-sensitive lateral flow immunoassay (LFIA) for rapid detection of potato leafroll virus (PLRV), an infectious agent of one of the most widespread potato diseases. The proposed LFIA combines the formation of sandwich immune complexes with gold nanoparticles (GNP) as labels and silver enhancement. The enhancement stage was realized using mixture of silver lactate and hydroquinone and subsequent addition of chloride-containing buffer to stop silver reduction. LFIA with silver enhancement was 15 times more sensitive (detection limit 0.2 ng/mL; 15 min) compared with conventional LFIA (detection limit 3 ng/mL; 10 min). The enhanced LFIA detected PLRV in leaves’ extracts of infected potato in dilutions higher than enzyme-linked immunosorbent assay. [ABSTRACT FROM AUTHOR]

Immunogold labeling of permeabilized whole‐mount cells or thin‐sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3‐dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation—but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze‐substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre‐embedding NANOGOLD‐silver immunocytochemistry. So obtained thin and semi‐thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context. [ABSTRACT FROM AUTHOR]

Lateral flow immunoassay (LFIA) is a widely used express method and offers advantages such as a short analysis time, simplicity of testing and result evaluation. However, an LFIA based on gold nanospheres lacks the desired sensitivity, thereby limiting its wide applications. In this study, spherical nanogold labels along with new types of nanogold labels such as gold nanopopcorns and nanostars were prepared, characterized, and applied for LFIA of model protein antigen procalcitonin. It was found that the label with a structure close to spherical provided more uniform distribution of specific antibodies on its surface, indicative of its suitability for this type of analysis. LFIA using gold nanopopcorns as a label allowed procalcitonin detection over a linear range of 0.5-10 ng mL with the limit of detection of 0.1 ng mL, which was fivefold higher than the sensitivity of the assay with gold nanospheres. Another approach to improve the sensitivity of the assay included the silver enhancement method, which was used to compare the amplification of LFIA for procalcitonin detection. The sensitivity of procalcitonin determination by this method was 10 times better the sensitivity of the conventional LFIA with gold nanosphere as a label. The proposed approach of LFIA based on gold nanopopcorns improved the detection sensitivity without additional steps and prevented the increased consumption of specific reagents (antibodies). [ABSTRACT FROM AUTHOR]

This paper addresses two key challenges toward an integrated forward error-correcting biosensor based on our previously reported self-assembled quick-response (QR) code. The first challenge involves the choice of the paper substrate for printing and self-assembling the QR code. We have compared four different substrates that includes regular printing paper, Whatman filter paper, nitrocellulose membrane and lab synthesized bacterial cellulose. We report that out of the four substrates bacterial cellulose outperforms the others in terms of probe (gold nanorods) and ink retention capability. The second challenge involves remote activation of the analyte sampling and the QR code self-assembly process. In this paper, we use light as a trigger signal and a graphite layer as a light-absorbing material. The resulting change in temperature due to infrared absorption leads to a temperature gradient that then exerts a diffusive force driving the analyte toward the regions of self-assembly. The working principle has been verified in this paper using assembled biosensor prototypes where we demonstrate higher sample flow rate due to light induced thermal gradients. [ABSTRACT FROM AUTHOR]

Cortisol is known as a stress biomarker. The measurement of cortisol levels is an early warning indicator for health conditions and diagnosis of stress-related diseases. Herein, a lateral flow immunoassay using a gold nanoparticle label with a silver enhancement system was developed for the simple, sensitive and rapid detection of cortisol. The developed assay was based on a competitive platform of which cortisol-BSA conjugate was immobilized at the test zone to compete with an analyte. The quantitative analysis was performed using gold nanoparticles (AuNPs) as signal labeling. Sequentially, the silver enhancement solution was applied in order to enhance the sensitivity of the assay with the results easily seen by the naked eye. Using this system, the limit of detection (LOD) was found to be 0.5 ng/mL with a 3.6 fold more sensitive detection than without the enhancement system (LOD = 1.8 ng/mL). The salivary cortisol analysis was in the range of 0.5-150 ng/mL (R2 = 0.9984), which is in the clinical acceptable range. For the semi-quantitative analysis, the intensity color of the results was analyzed using an image processing program. The proposed method was successfully applied to detect cortisol in saliva. In addition, the results from our method also complied with the ones of those obtained by using the commercial enzyme-linked immunosorbent assay (ELISA). This developed assay offers great promise for a non-invasive screening test of salivary cortisol. [ABSTRACT FROM AUTHOR]

A sensitive and rapid absorbance based immunosensor that utilizes ex situ functionalized porous silica monoliths as volumetric optical detection elements are demonstrated in this study. The porous monolith structure facilitates high capture probe density and short diffusion length scales, enabling sensitive and rapid assays. Silica monoliths, synthesized and functionalized with immunocapture probes off-chip before integration into a sealed thermoplastic microfluidic device, serve to capture target antigens during perfusion through the porous structure. Gold nanoparticle immunoconjugates are combined with silver enhancement to create microscale silver clusters, followed by perfusion of an aqueous sucrose solution to limit light scattering and enhance optical signal. Using this approach, detection limits as low as 1 ng/mL are achieved for a sandwich assay, with a dynamic range of at least 4 logs. The results confirm that the combination of on-chip index matching with functionalized porous silica monoliths can enable simple and practical flow-through immunoassays for the sensitive and rapid detection of target antigens. [ABSTRACT FROM AUTHOR]

Despite the simplicity and usability of gold nanoparticle (AuNP)-based colorimetry in a large panel of bioassays, this technique still suffers from a poor detection limit. To overcome this issue, we report an improved AuNP-based colorimetric assay in combination with simple centrifugation and silver enhancement. As a proof-of-concept, two types of enzymes (i.e., protein phosphatase and protease) were constructed based on the self-assembly of thiol-stabilized AuNPs in the presence of peptides and metal ions. When the AuNP solutions were subjected to a short cycle of centrifugation and silver enhancement, the signal-to-background ratio (SBR) was distinctly improved by a factor of 5–10 in both enzyme activity assays, as compared to that of AuNP-based colorimetry. The detection limit achieved by using the silver enhancement was determined to be improved by a factor of 1.0–3.4. Furthermore, grayscale images of the silver enhancement allowed for a rapid and simple enzyme assay that did not require measuring the absorbance. Due to its ability to improve the detection senstivity in a facile way, we anticipate that this approach will be suitable for use in many AuNP colorimetric assays. [ABSTRACT FROM AUTHOR]

Electrochemical DNA sensors represent a simple, accurate and economical platform for DNA detection. Gold nanoparticles are known to be efficient labels in electrochemical sensors and to be viable materials to modify the surface of electrodes thereby to enhance the detection limit of the sensor. For surface modification, gold nanoparticles are used in combination with nanomaterials like graphene, graphene oxide, or carbon nanotubes to improve electrochemical performance in general. This review (with 116 refs.) mainly covers the advances made in recent years in the use of gold nanoparticles in DNA sensing. It is divided into the following main sections: (a) An introduction covers aspects of electrochemical sensing of DNA and of appropriate nanomaterials in general. (b) The use of gold nanoparticles in DNA is specifically addressed next, with subsections on AuNPs acting as electrochemical labels, electron transfer mediators, signal amplifiers, carriers of electroactive molecules, catalysts, immobilization platforms, on silver enhancement strategies, on AuNPs modified with carbonaceous materials (such as graphenes and nanotubes), and on multiple amplification schemes. The review concludes with a discussion of current challenges and trends in terms of highly sensitive DNA based sensing using AuNPs. [ABSTRACT FROM AUTHOR]

A rapid, visual protein detection method of human immunoglobulin E (hIgE) was developed for 96-well microplate arrays by using aptamer modifying silver nanoparticles. An aptamer specifically recognizing IgE and biotinylated oligonucleotides were used to modify AgNPs and the resulting nanoprobes were employed to assay model analyte hIgE. When addition of the analyte as well as the probes to the microplate array on which Goat anti-human IgE was first immobilized, the signal of this “sandwich” format was further amplified by silver enhancement technique based on silver ions reductive reagents and achieved a visual dots in the microplates. To achieve quantitative analysis, a series of gradient concentration streptavidin (SA) was immobilized in the same well. As a result, an internal standard curve was plotted by the signal of different concentration of SA based on streptavidin-biotin coupling reaction. With the help of this curve, a quantitative detection of IgE was established and a detection limit of 20 ng mL −1 could be obtained. The presented method was further carried out for detection of human serum samples and relative consistent results were found. [ABSTRACT FROM AUTHOR]

This work demonstrated the determination of proteolytically active prostate specific antigen (paPSA), a potential biomarker for prostate cancer diagnosis, in human serum using a sensitive and low-cost electrochemical sensor. A specifically designed peptide probe was immobilized on the surface of a 96-well plate. The probe could be recognized by paPSA causing cleavage of the peptide, resulting in a decrease in the thiols group remaining on the probe. Gold nanoparticles (AuNPs) were attached to the peptide thiol groups by self-assembly. Hence the amount of AuNPs relates to the length of peptide probe. After cleavage and binding of AuNPs, an amplification step was performed using a silver enhancer solution. The quantity of deposited silver was then measured by differential pulse anodic stripping voltammetry (DPASV) using a disposable screen-printed carbon electrode (SPCE). The signal for paPSA detection was the linear range from 0.1 to 100 ng mL − 1 , with a detection limit of 27 pg mL − 1 . We also showed that the assay was reliable and has potential for clinical applications. [ABSTRACT FROM AUTHOR]

A highly sensitive and selective electrochemical DNA biosensor for detection of Ag + was developed based on amplification of DNA–Au bio–bar codes and silver enhancement. Cytosine-rich single stranded DNA oligonucleotide (sub–DNA) was immobilized on the surface of the gold electrode via Au–S chemical interaction. In the presence of Ag + , with the interaction of cytosine–Ag + –cytosine (C–Ag + –C), cytosine-rich DNA oligonucleotide probes-labeled AuNPs have a hybridization with sub–DNA and then form a duplex-like structure, following the AuNPs-catalyzed silver enhancement. The whole detection procedure of Ag + based on the developed biosensor was evaluated by electrochemical techniques. Owing to the hybridization of sandwich assay format and distinct electrochemical signal amplification of silver enhancement, the proposed biosensors exhibited a good analytical performance for sensitive transduction of Ag + recognition with a wide detection linear range from 5 pM to 50 μM, and a low detection limit of 2 pM. The developed biosensor was highly selective and its practical applicability in real water samples was also tested with satisfactory results. [ABSTRACT FROM AUTHOR]

In this paper, we report a sensitive, simple and inexpensive analytical method for the immunoassay microarray based on a smartphone in which various harmful substances in milk could be assayed. Tetracyclines (TCs) and Quinolones (QNs) were selected as the model targets in this study. TCs and QNs antigens were immobilized in the microarray and then samples containing free of antibiotics and corresponding antibodies as well as AgNPs labeled secondary antibodies were added to the microarray. The signal of this competitive format was further amplified by silver enhancement technique based on the development reagents and achieved a visual dots in the array. The resulting microarray could be detected by the smartphone placed in the minicartridge. The limit of detection (LOD) of this novel detection platform was 1.51 ng mL −1 (TCs) and 1.74 ng mL −1 (QNs). To achieve one-well quantitative analysis, a series of gradient concentration mouse IgG was immobilized in the same well. As a result, an internal standard curve was plotted by the signal of different concentrations of mouse IgG. The results showed that a quantitative detection of TCs and QNs established were consistent with external standard curve. Compared to other methods, this method was superior in terms of detection limit, time saving, and one-well quantitative detected with smartphone which were simple sample-preparation. [ABSTRACT FROM AUTHOR]

Abstract Silver-enhanced labeling method has been employed in immunochromatographic assays for improving the sensitivity of detecting pathogens. In this paper, we apply the silver enhancement technique for biomolecular signal amplification in a gold nanoparticle-based conductimetric biochip. We show that the response of the silver-enhanced biochip comprises two distinct regions namely: (a) a sub-threshold region where conduction occurs due to electron hopping between silver islands and the electrolyte and (b) an above-threshold region where the conduction is due to a direct flow of electrons. These two regions are characterized by different conduction slopes, and we show that combining the information from both these regions can improve the sensitivity of the biochip. Results from fabricated prototypes show a dynamic range of more than 40 dB and with a detection limit less than 240 pg/mL. The fabrication of the biochip is compatible with standard complementary metal–oxide–semiconductor (CMOS) processes making it ideal for integration in next-generation CMOS biosensors.

The authors describe a method for highly sensitive and selective determination of the activity of protein kinase (PKA). It is based on the finding that silver nanoclusters (AgNCs) can act as a nucleus to catalyze further deposition of silver nanoparticles. This causes the color of a solution to change from pale yellow to black. In the detection scheme presented here, the substrate peptide is phosphorylated by PKA in the presence of ATP. The resulting phosphopeptides bind to oligonucleotide-stabilized AgNCs in the presence of Zr(IV) ions due to electrostatic interactions between Zr(IV) and the phosphate groups, thereby capping the AgNCs. The silver enhancement process (leading to a color change to black) does not work if the AgNCs are capped. The degree of inhibition is proportional to the activity of the kinase. The color change can be detected visually or photographically in a microplate format by exploiting the changes in the grey values of the digital photos. In addition, the DNA-AgNCs display fluorescence emission at 635 nm when excited at 565 nm. Electrochemical assays were performed (at a working voltage as low as 38 mV vs. Ag/AgCl) by using a glassy carbon electrode modified with a solution containing AgNCs, Zr(IV) ions and the peptide, and immersing it into the silver enhancement solution. The assay is highly sensitive and selective. It was applied to the determination of PKA in lysates of HeLa cells. The detection limits typically are between 32 and 37 U⋅ L based on a signal-to-noise ratio of 3. [ABSTRACT FROM AUTHOR]

A sensitive and rapid impedemetric immunosensor is demonstrated utilizing porous volumetric microfluidic detection elements and silver enhanced gold nanoparticle probes. The porous detection elements significantly increase capture probe density and decrease diffusion length scales compared to conventional planar sensors to improve target capture efficiency and enhance impedance signal. In this work, a packed bed of silica beads functionalized with antibody probes serves as a porous sensor element within a thermoplastic microchannel, with an interdigitated gold electrode microarray used to measure impedance changes caused by the concentration dependent formation of silver aggregates. The measured impedance change is independent of electrode spacing, enabling a device with low resolution electrodes to achieve a sandwich immunoassay detection limit between 1 and 10 ng/mL with a 4-log dynamic range, with a total assay time of 75 min. [ABSTRACT FROM AUTHOR]