Highly sensitive protein detection using recombinant spores and lateral flow immunoassay.

Lateral flow immunoassays (LFIs) can be used to detect intact bacteria or spores; when gold nanoparticles (AuNPs) are used as the signal reporters, the detection limits are very low. Spore-based surface display has been widely studied for enzyme immobilization and live-nontoxic oral vaccines. In this study, recombinant spores were used to improve the sensitivity of a LFI. We developed a test kit that combines streptavidin-displayed spores with a LFI assay for rapid protein detection. The recombinant spores served as a signal amplifier and AuNPs were used as the signal reporters. For detection of ß-galactosidase, which was used as the model protein, the detection limit was about 10-15 mol, while that of the conventional LFI is about 10-12 mol. In both methods, nanogold was used as the colorimetric signal and could be observed with the naked eye. This method improved LFI sensitivity without sacrificing its advantages. Furthermore, enhanced green fluorescent protein (eGFP) was also displayed on the surface of the streptavidin-displayed spores. Without AuNPs, the fluorescent recombinant spores acted as the signal, which could be detected by a fluorescence detector, such as a fluorescence microscope. The detection limit was 10-16 mol under fluorescence microscopy whose magnification was 25-fold. Therefore, in conclusion, in this proof of concept study, the detection limits of both proposed methods were far superior to those of traditional LFI assay.

Detection of biotin with zeptomole sensitivity using recombinant spores and a competition assay.

Detection of protein-binding analytes is important for many applications. Currently, various instrument-based techniques are used for detecting protein-binding analytes. However, such techniques have several limitations including high cost and time-consuming sample processing. In order to overcome these limitations, we developed a sensitive competition assay for the detection of protein-binding analytes using recombinant endospores as a sensing element. The method is based on the competition between the biotin, the model analyte, and a biotin-magnetic bead complex to bind the recombinant spores containing the biotin binding region of streptavidin. After magnetic attraction, the residual spores in the suspension are spread on plates to form colonies which are used to count the amount of the residual spores; the higher the residual ratio of spores, the more biotin in the samples. The linear range was from 150 zmol to 1.5 fmol and the limit of detection of the assay was 150 zmol. The assay proposed herein is sensitive and does not require any expensive equipment. It is suitable for qualitative or semi-quantitative analysis such as screening tests for the detection of toxic chemicals.

Dichroic Behavior of Gold Nanoparticles Synthesized in Aqueous Solution with Insufficient Reducing Agent.

The spherical gold nanoparticles (AuNPs) typically are red in solution. However, in this study, the dichroic and spherical AuNPs were synthesized using a modified seeding growth method under reducing agent insufficiency in an aqueous solution. This particular AuNP solution is orange in reflected light and red in transmitted light. The reflectance curves confirm that the dichroic AuNPs are different from the classic AuNPs. With particle assembling, the AuNP solution is fainter orange in reflected light, but purple in transmitted light when the color of classic spherical AuNP solution is purple in both lights. Furthermore, the aggregated-nanogold solutions were added to HAuCl4 solutions with the addition of an insufficient amount reducing agent. The solution changed from faint orange to bright orange in reflected light and from purple to blue in transmitted light. It indicates that the gold assembling under a reducing agent insufficiency, not the shape of AuNP, causes the dichroic phenomenon. To the best of our knowing, this is the first study to report how the AuNP is synthesized, not the shape, affects the color of the AuNP.

Enhancing antibody detection sensitivity in lateral flow immunoassays using endospores of Bacillus subtilis as signal amplifiers.

Antibody detection is the critical first step for tracking the spread of many diseases including COVID-19. Lateral flow immunoassay (LFIA) is the most commonly used method for rapid antibody detection because it is easy-to-use and inexpensive. However, LFIA has limited sensitivity when gold nanoparticles (AuNPs) are used as the signals. In this study, the endospores of Bacillus subtilis were used in combination with AuNP in a LFIA to detect antibodies. The endospores serve as a signal amplifier. The detection limit was about 10-8 M for anti-beta galactosidase antibody detection whereas the detection limit of conventional LFIA is about 10-6 M. Furthermore, the proposed methods have no additional user steps compared with the traditional LFIA. This method, therefore, improved the sensitivity 100-fold without compromising any advantages of LFIA. We believe that the proposed method will be useful for detection of antibodies against HIV, Zika virus, SARS-CoV-2, and so on.

Micro-RNA quantification using DNA polymerase and pyrophosphate quantification.

A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20-100 µg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/µg RNA and 0.71 fmol/µg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays.

3D Printing of Collagen/Oligomeric Proanthocyanidin/Oxidized Hyaluronic Acid Composite Scaffolds for Articular Cartilage Repair.

Articular cartilage defects affect millions of people worldwide, including children, adolescents, and adults. Progressive wear and tear of articular cartilage can lead to progressive tissue loss, further exposing the bony ends and leaving them unprotected, which may ultimately cause osteoarthritis (degenerative joint disease). Unlike other self-repairing tissues, cartilage has a low regenerative capacity; once injured, the cartilage is much more difficult to heal. Consequently, developing methods to repair this defect remains a challenge in clinical practice. In recent years, tissue engineering applications have employed the use of three-dimensional (3D) porous scaffolds for growing cells to regenerate damaged cartilage. However, these scaffolds are mainly chemically synthesized polymers or are crosslinked using organic solvents. Utilizing 3D printing technologies to prepare biodegradable natural composite scaffolds could replace chemically synthesized polymers with more natural polymers or low-toxicity crosslinkers. In this study, collagen/oligomeric proanthocyanidin/oxidized hyaluronic acid composite scaffolds showing high biocompatibility and excellent mechanical properties were prepared. The compressive strengths of the scaffolds were between 0.25-0.55 MPa. Cell viability of the 3D scaffolds reached up to 90%, which indicates that they are favorable surfaces for the deposition of apatite. An in vivo test was performed using the Sprague Dawley (SD) rat skull model. Histological images revealed signs of angiogenesis and new bone formation. Therefore, 3D collagen-based scaffolds can be used as potential candidates for articular cartilage repair.

Dose-dependent effect of Lactobacillus rhamnosus on quantitative reduction of faecal rotavirus shedding in children.

Beneficial effects of probiotics in acute infectious diarrhoea in children are mainly seen in watery diarrhoea and viral gastroenteritis. Lactobacillus rhamnosus, one the most extensively studied probiotic strains, is effective in shortening courses of acute diarrhoea in children. However, the dose-dependent effect of Lactobacillus upon quantification of faecal rotavirus shedding in humans remains little known. Thus, an open-label randomized trial in 23 children with acute rotaviral gastroenteritis was undertaken by randomly allocating patients to receive one of the three regimens for 3 days: daily Lactobacillus rhamnosus 35 (Lcr35) with 0 CFU/day to six patients in the control group, 2 x 10(8) CFU/day to nine patients in the low-dose group, and 6 x 10(8) CFU/day to eight patients in the high-dose group. Faecal samples were collected before and after the 3-day regimen for measurements of rotavirus concentrations by ELISA. There was no statistically significant change in faecal rotavirus concentrations in either the control group (119.2 x 10(5) particles/ml vs. 23.7 x 10(5) particles/ml, p = 0.075) or the low-dose group (36.1 x 10(5) particles/ml vs. 73.5 x 10(5) particles/ml, p = 0.859). However, the high-dose group had a significant reduction of faecal rotavirus concentration (64.2 x 10(5) particles/ml vs. 9.0 x 10(5) particles/ml, p = 0.012). Without any exception, the faecal rotavirus concentrations of all eight patients in the high-dose Lcr35 group declined by 86% after 3 days when compared with those before Lcr35 administration. In conclusion, this is the first report to provide quantitative evidence of the dose-dependent effect of Lactobacillus rhamnosus, a minimal effective dose of 6 x 10(8) CFU for 3 days, upon the faecal rotavirus shedding in paediatric patients.

A competitive immunoassay for biotin detection using magnetic beads and gold nanoparticle probes.

Haptens are small molecules with low molecular weight that include biotin and many toxins in food. In this study, we used biotin as a model molecule for hapten detection. In this competitive immunoassay anti-biotin antibody-modified magnetic beads (Ab-MBs) and biotinylated thiol-DNA gold nanoparticles (biotin-GNPs) were used. The assay contains three reactions, the mixing of the sample and Ab-MBs, the capture of biotin-GNPs by Ab-MBs and the magnetic attraction. When biotin molecules were absent, the solution was transparent because biotin-GNPs bound to Ab-MBs which were caught by an external magnetic field. When biotin was present, the supernatant was red because the Ab-MBs bound to the analytes and the gold nanoparticles were still in solution. It was possible to complete all the operating steps in 15 min. The limit of detection (LOD) was 2 pmol. This rapid competitive-immunoassay has potential for application in detection of other haptens.

In vivo toxicologic study of larger silica nanoparticles in mice.

Silica nanoparticles (SiNPs) are being studied and used for medical purposes. As nanotechnology grows rapidly, its biosafety and toxicity have frequently raised concerns. However, diverse results have been reported about the safety of SiNPs; several studies reported that smaller particles might exhibit toxic effects to some cell lines, and larger particles of 100 nm were reported to be genotoxic to the cocultured cells. Here, we investigated the in vivo toxicity of SiNPs of 150 nm in various dosages via intravenous administration in mice. The mice were observed for 14 days before blood examination and histopathological assay. All the mice survived and behaved normally after the administration of nanoparticles. No significant weight change was noted. Blood examinations showed no definite systemic dysfunction of organ systems. Histopathological studies of vital organs confirmed no SiNP-related adverse effects. We concluded that 150 nm SiNPs were biocompatible and safe for in vivo use in mice.

Salmonella detection using 16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay.

An ultrasensitive, simple, and fast lateral flow immunoassay for Salmonella detection using gold nanoparticles conjugated with a DNA probe, which is complementary to the 16S ribosomal RNA and DNA of Salmonella, has been developed. The detection limit is 5 fmol for the synthetic single-stranded DNA. For the Salmonella cultured samples, the nucleic acids from 10(7) bacteria were rapidly detected in 30 min. After silver enhancement, the detection limit was as low as 10(4) cells which is lower than 10(5) bacteria cells, the human infective dose of food-borne Salmonella. Furthermore, the probes used in this study are specific to Salmonella compared to several other Enterobacteriaceae. This approach would be a useful tool for microbial detection regarding food safety or clinical diagnosis. It is also suitable for large-scale screening in developing countries because it is low-cost, sensitive, specific and convenient.

Determination of copy number of short tandem repeat using NAD-dependent ligase and pyrosequencing-compatible method.

A sensitive and pyrosequencing-compatible method for determining the copy number of the short tandem repeat (STR) is presented in this study. When Escherichia coli ligase catalyzes the ligation of primer and probes complementary to the proper sites of the target DNA template, it converts nicotinamide adenine dinucleotide to adenosine monophosphate (AMP) and nicotinamide. The AMP release level is proportional to the copy number of the STR and can be measured using adenylate kinase, pyruvate kinase, and luciferase. Unlike current standard methods based on electrophoresis, the present assay is sensitive to the point mutation. Furthermore, after determination of the copy number of the tandem repeat using the proposed method, the DNA templates, primer and probes immobilized onto super paramagnetic beads can be washed and pyrosequencing can be applied for the remaining DNA sequencing. This assay is specially efficient to handle a large number of samples because massively parallel tests could be executed in a microplate photometer. Furthermore, it can work with the pyrosequencing for further sequencing like genome sequencing.

MicroRNA detection using lateral flow nucleic acid strips with gold nanoparticles.

In this study, the tested microRNA and the detection probe perfectly match with the capture probe instead of the traditional sandwich methods in which the tested oligonucleotide matches with the detection and capture probes. To avoid non-specific signals, mung-bean nuclease, a single-strand-specific nuclease, catalyzes the degradation of the capture probe if there is no tested miRNA in the samples. The gold nanoparticles conjugate the thiol-DNA as the detection probe and the biotin-single strand DNA serves as the capture probe. The avidin-biotin-Au-sample complex is captured by the anti-avidin antibody immobilized on a flow strip. The detection and quantification of the gold nanoparticle signal indicate the existence and quantity of the target miRNA. One fmol and five amol of the synthetic microRNA were detected without and with the silver enhancement, respectively. This highly sensitive and specific assay takes about 70 min after the RNA purification and preparation. It is simple, convenient, fast, and suitable for point-of-care.

Identification of Salmonella using colony-print and detection with antibody-coated gold nanoparticles.

In this study, we developed an easy screening test that identifies Salmonella in 2 h after colony-print which is a procedure based on the transfer of surface cells of the colonies to a nitrocellulose membrane. Gold nanoparticles coated with the anti-Salmonella antibody were used to highlight the Salmonella spp. on the membrane to facilitate the selectivity. On Hektoen agar, 134 stool samples containing black or crystalloid colonies were identified using the proposed method. Without any equipment, such as microscope, the red dots corresponding Salmonella were observed. After colony-print test, 22 of the isolates were correctly identified as Salmonella to achieve 100% sensitivity. 111 samples were correctly identified as non-Salmonella spp., but one was incorrectly identified as Salmonella. The specificity is 99.1%. This method is simple, straightforward, inexpensive, and fast. It can be easily applied to the routine workload of clinical laboratories, and can be very useful when large amounts of fecal samples should be evaluated for rapid screening and diagnosis.

Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases.

The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly.

Detection of specific bioparticles using antibodies-coated gold nanoparticles and dielectrophoretic impedance measurement.

In this study, we developed an easy and quick test capable of identifying specific bacteria in one hour. The protocol was established based on the measurement of bacteria quantity on a biochip with comb-like electrodes. Gold nanoparticles coated with anti-Salmonella antibody were used to enhance the dielectrophretic property of Salmonella spp. on the biochip to facilitate the sensitivity.

Development of a biochip using antibody-coated gold nanoparticles to detect specific bioparticles.

This study developed a method of detecting bioparticles such as Salmonella that exist in the biological samples. The method employed a substrate with interlaced comb-like electrodes into which the mixtures of biological samples and antibody-coated gold nanoparticles were added. The alternative signals with appropriate frequency bands were then conducted into the comb-like electrodes to change the dielectrophoresis force. The gold-modified Salmonella can be adsorbed on the edges of the electrodes and isolated from various biological samples. The impedance of the adsorbed Salmonella on the edges of the electrodes was measured and comparison of the impedance between the electrodes with and without Salmonella can quantify the amount of the adsorbed Salmonella.

Development of zeptomole and attomolar detection sensitivity of biotin-peptide using a dot-blot gold nanoparticle immunoassay.

An ultrasensitive, simple, and fast immunoassay for biotin-peptide detection using gold nanoparticles conjugated with antibodies has been developed. Biotin was covalently attached to a peptide and the biotin-peptide bound on a nitrocellulose membrane. Antibody-coated gold nanoparticles bound to the biotin-peptide formed red dots. With this method, 100 amol of the biotin-peptide was detected and no immunogold was bound to the membrane in the absence of biotin. The relative intensity of each dot was scored using Quantity One, a quantitative analysis software program. The linear working range of this assay was between 1 pmol and 1 micromol. The assay sensitivity was increased by silver enhancement to 100 zmol, and the linear working range was between 100 zmol and 100 fmol. This assay can be extended to detect target molecules, such as dioxin, digoxin, mercury, and so on, with matched antibodies and has potential broad applications in immunoassay.