Single-Domain Antibody-Gold Nanoparticle Bioconjugates as Immunosensors for the Detection of Hantaviruses.

INTRODUCTION: Hantavirus, a zoonotic pathogen, causes severe syndromes like hemorrhagic fever with renal syndrome (HFRS), sometimes fatal in humans. Considering the importance of detecting the hantavirus antigen, the construction of an immunosensor is essential. The structural and functional characteristics of camelid nanobodies (VHHs) encourage their application in the areas of nanobiotechnology, therapeutics, diagnostics, and basic research. Therefore, this study aimed to standardize stable bioconjugates using gold nanoparticles (AuNPs) and VHHs, in order to develop immunobiosensors for the diagnosis of hantavirus infection. METHODS: Immobilized metal affinity chromatography (IMAC) was performed to obtain purified recombinant anti-hantavirus nucleocapsid nanobodies (anti-prNΔ85 VHH), while AuNPs were synthesized for bioconjugation. UV-visible spectrophotometry and transmission electron microscopy (TEM) analysis were employed to characterize AuNPs. RESULTS: The bioconjugation stability parameters (VHH-AuNPs), analyzed by spectrophotometry, showed that the ideal pH value and VHH concentration were obtained at 7.4 and 50 µg/mL, respectively, after addition of 1 M NaCl, which induces AuNP aggregation. TEM performed before and after bioconjugation showed uniform, homogeneous, well-dispersed, and spherical AuNPs with an average diameter of ~ 14 ± 0.57 nm. Furthermore, high-resolution images revealed a thin white halo on the surface of the AuNPs, indicating the coating of the AuNPs with protein. A biosensor simulation test (dot blot-like [DB-like]) was performed in stationary phase to verify the binding and detection limits of the recombinant nucleocapsid protein from the Araucária hantavirus strain (prN∆85). DISCUSSION: Using AuNPs/VHH bioconjugates, a specific interaction was detected between 5 and 10 min of reaction in a dose-dependent manner. It was observed that this test was sensitive enough to detect prNΔ85 at concentrations up to 25 ng/µL. Considering that nanostructured biological systems such as antibodies conjugated with AuNPs are useful tools for the development of chemical and biological sensors, the stability of the bioconjugate indicates proficiency in detecting antigens. The experimental results obtained will be used in a future immunospot assay or lateral flow immunochromatography analysis for hantavirus detection.

Single domain antibodies in the development of immunosensors for diagnostics.

Scientific advances in nanotechnology and nanoscience have enabled stability optimization and signal amplification in immunoassays by taking advantage of unique properties of nanomaterials. Biosensors based on antibodies and their fragments, also called immunosensors, are compact tools capable of providing refined antigen detection capacity. Different immunoassays that utilize these molecules for biorecognition have been used as diagnostic tools. In this regard, camelid single domain antibodies fulfill several requirements, such as nanometric size, high affinity, specificity, solubility, stability, biotechnological versatility, and low cost of production, constituting an important source for the development of immunodiagnostic devices. In this review, the main technological advances involving this specific class of molecules, as well as their major biotechnological applications will be addressed, with emphasis on their use as biosensors applied to diagnostics in human health.

Antimicrobial susceptibility assays in paper-based portable culture devices.

To detect antibiotic-resistant bacteria in areas remote from microbiology laboratories, we designed portable culture devices performing an analogue of the Kirby-Bauer disk diffusion test inside patterned papers embedded in tape. We quantified the antibiotic susceptibility of several strains of Escherichia coli and Salmonella typhimurium by measuring blue-colored zones of inhibited growth.

Inactivation of Alicyclobacillus acidoterrestris in orange juice by saponin extracts combined with heat-treatment.

Alicyclobacillus acidoterrestris is a spoilage-causing bacterium in fruit juices. The inactivation of this bacterium by commercial saponin and saponin purified extract from Sapindus saponaria fruits combined with heat-treatment is described. We investigated heat treatment (87, 90, 95, and 99°C) with incubation time ranging from 0 to 50min, in both concentrated and reconstituted juice. Juices were inoculated with 1.0×10(4)CFU/mL of A. acidoterrestris spores for the evaluation of the best temperature for inactivation. For the temperatures of 87, 90, and 95°C counts of cell viability decreased rapidly within the first 10 to 20min of incubation in both concentrated and reconstituted juices; inactivation at 99°C ensued within 1 and 2min. Combination of commercial saponin (100mg/L) with a very short incubation time (1min) at 99°C showed a reduction of 2.34 log cycle for concentrated juice A. acidoterrestris spores (1.0×10(4)CFU/mL) in the first 24h of incubation after treatments. The most efficient treatment was reached with 300, 400 or 500mg/L of purified extract of saponins from S. saponaria after 5days of incubation in concentrated juice, and after 5days with 300 and 400mg/L or 72h with 500mg/L in reconstituted juice. Commercial saponin and purified extracts from S. saponaria had similar inactivation power on A. acidoterrestris spores, without significant differences (P>0.05). Therefore, purified extract of saponins can be an alternative for the control of A. acidoterrestris in fruit juices.

Portable self-contained cultures for phage and bacteria made of paper and tape.

In this paper, we demonstrate that a functional, portable device for the growth of bacteria or amplification of bacteriophage can be created using simple materials. These devices are comprised of packing tape, sheets of paper patterned by hydrophobic printer ink, and a polydimethyl siloxane (PDMS) membrane, which is selectively permeable to oxygen but non-permeable to water. These devices supply bacteria with oxygen and prevent the evaporation of media for a period over 48 h. The division time of E. coli and the amplification of the phage M13 in this device are similar to the rates measured on agar plates and in shaking cultures. The growth of bacteria with a fluorescent mCherry reporter can be quantified using a flatbed scanner or a cell phone camera. Permeating devices with commercial viability dye (PrestoBlue) can be used to detect low copy number of E. coli (1-10 CFU in 100 µL) and visualize microorganisms in environmental samples. The platform, equipped with bacteria that carry inducible mCherry reporter could also be used to quantify the concentration of the inducer (here, arabinose). Identical culture platforms can, potentially, be used to quantify the induction of gene expression by an engineered phage or by synthetic transcriptional regulators that respond to clinically relevant molecules. The majority of measurement and fabrication procedures presented in this report have been replicated by low-skilled personnel (high-school students) in a low-resource environment (high-school classroom). The fabrication and performance of the device have also been tested in a low-resource laboratory setting by researchers in Nairobi, Kenya. Accordingly, this platform can be used as both an educational tool and as a diagnostic tool in low-resource environments worldwide.

A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis.

In this paper we compare the effects of three representative PCR inhibitors using quantitative PCR (qPCR) and multiplex STR amplification in order to determine the effect of inhibitor concentration on allele dropout and to develop better ways to interpret forensic DNA data. We have used humic acid, collagen and calcium phosphate at different concentrations to evaluate the profiles of alleles inhibited in these amplifications. These data were correlated with previously obtained results from quantitative PCR including melt curve effects, efficiency changes and cycle threshold (Ct) values. Overall, the data show that there are two competing processes that result from PCR inhibition. The first process is a general loss of larger alleles. This appears to occur with all inhibitors. The second process is more sequence specific and occurs when the inhibitor binds DNA, altering the cycle threshold and the melt curve. This sequence-specific inhibition results in patterns of allele loss that occur in addition to the overall loss of larger alleles. The data demonstrate the applicability of utilizing real-time PCR results to predict the presence of certain types of PCR inhibition in STR analysis.

Sequence-specific electrochemical detection of Alicyclobacillus acidoterrestris DNA using electroconductive polymer-modified fluorine tin oxide electrodes.

This study outlines the quantification of low levels of Alicyclobacillus acidoterrestris in pure cultures, since this bacterium is not inactivated by pasteurization and may remain in industrialized foods and beverages. Electroconductive polymer-modified fluorine tin oxide (FTO) electrodes and multiple nanoparticle labels were used for biosensing. The detection of A. acidoterrestris in pure cultures was performed by reverse transcription polymerase chain reaction (RT-PCR) and the sensitivity was further increased by asymmetric nested RT-PCR using electrochemical detection for quantification of the amplicon. The quantification of nested RT-PCR products by Ag/Au-based electrochemical detection was able to detect 2 colony forming units per mL (CFU mL(-1)) of spores in pure culture and low detection and quantification limits (7.07 and 23.6 nM, respectively) were obtained for the target A. acidoterrestris on the electrochemical detection bioassay.

Semiquantitative determination of Alicyclobacillus acidoterrestris in orange juice by reverse-transcriptase polymerase chain reaction and capillary electrophoresis--laser induced fluorescence using microchip technology.

The semiquantitative detection of Alicyclobacillus acidoterrrestris in orange juice by reverse-transcriptase polymerase chain reaction (RT-PCR) with a linear dynamic range of 2 x 10(5)-2 colony forming units (CFU)/mL in terms of cell count is described. Separation, detection, and quantification of the RT-PCR products were accomplished using the Agilent 2100 bioanalyzer in conjunction with the DNA 1000 LabChip kit. After 0 and 12 h of enrichment, it was possible to generate a linear standard curve between the amount of cells and amplicon concentration of RT-PCR and PCR products. Using this method, cell diminution was verified in samples of orange juice treated with a natural inhibitor (Sapindus saponaria), determining the persistence of viable cells. Semiquantitative RT-PCR using the Agilent 2100 bioanalyzer is a potentially useful approach for rapid in vitro determination of A. acidoterrestris and monitoring of inhibitor susceptibility for the orange juice-producing industry.