Colorimetric bio-barcode amplification assay for cytokines

The bio-barcode amplification assay has become a powerful tool in detecting tens to hundreds of biological targets such as proteins and nucleic acids in the entire sample. However, current bio-barcode detection schemes still require many experimental steps including microarrayer-based immobilization of oligonucleotides on a glass chip, silver enhancement of immobilized gold nanoparticles on a chip, and light-scattering measurement. Here, we report a colorimetric bio-barcode method that minimizes the above requirements while detecting 30 aM concentrations of cytokines (approximately 3 orders of magnitude more sensitive than conventional nonenzymatic cytokine detection assays). The assay is based on porous microparticles, which enable loading of a large number of barcode DNA per particle, and gold nanoparticle-based colorimetric barcode detection method.     

Fluoroimmunoassay for antigen based on fluorescence quenching signal of gold nanoparticles

A unique, sensitive, and highly specific fluoroimmunoassay system for antigen detection using gold and magnetic nanoparticles has been developed. The assay is based on the fluorescence quenching of fluorescein isothiocyanate caused by gold nanoparticles coated with monoclonal antibody. To demonstrate its analytical capabilities, the magnetic nanoparticles were coated with anti-alpha-fetoprotein polyclonal antibodies, which specifically bound with alpha-fetoprotein. Gold nanoparticles coated with anti-alpha-fetoprotein monoclonal antibodies could sandwich the alpha-fetoprotein captured by the magnetic nanoparticle probes. The sandwich-type immunocomplex was formed on the surface of magnetic nanoparticles and could be separated by a magnetic field. The supernatant liquid, which contained the unbound gold nanoparticle probes, was used to quench the fluorescence, and the fluorescence intensity of fluorescein isothiocyanate at 516 nm was proportional to the alpha-fetoprotein concentration. The result showed that the limit of detection of alpha-fetoprotein was 0.17 nM. This new system can be extended to detect target molecules with matched antibodies and has broad potential applications in immunoassay and disease diagnosis.     

Improving the signal sensitivity and photostability of DNA hybridizations on microarrays by using dye-doped core-shell silica nanoparticles

The development of new highly sensitive and selective methods for microarray-based analysis is a great challenge because, for many bioassays, the amount of genetic material available for analysis is extremely limited. Currently, imaging and detection of DNA microarrays are based primarily on the use of organic dyes. To overcome the problems of photobleaching and low signal intensities of organic dyes, we developed a new class of silica core-shell nanoparticles that encapsulated with cyanine dyes and applied the dye-doped nanoparticles as labeling in the DNA microarray-based bioanalysis. The developed nanoparticles have core-shell structure containing 15-nm Au colloidal cores with 95 dye-alkanethiol (dT)20 oligomers chemisorbed on the each Au particle surface and 10-15-nm silica coatings bearing thiol functional groups. To be utilized for microarray detection, the dye-doped nanoparticles were conjugated with DNA signaling probes by using heterobifunctional cross-linker. The prepared nanoparticle conjugates are stable in both aqueous electrolytes and organic solvents. Two-color DNA microarray-based detection was demonstrated in this work by using Cy3- and Cy5-doped nanoparticles in sandwich hybridization. The use of the fluorophore-doped nanoparticles in high-throughput microarray detection reveals higher sensitivity with a detection limit of 1 pM for target DNA in sandwich hybridization and greater photostable signals than the direct use of organic fluorophore as labeling. A wide dynamic range of approximately 4 orders of magnitude was also found when the dye-doped nanoparticles were applied in microarray-based DNA bioanalysis. In addition, the use of these dye-doped nanoparticles as the labeling in hybridization also improved the differentiation of single-nucleotide polymorphisms. This work offers promising prospects for applying dye-doped nanoparticles as labeling for gene profiling based on DNA microarray technology.     

Combination of functionalized nanoparticles and polymerase chain reaction-based method for SARS-CoV gene detection

Rapid and sensitive detection of SARS associated coronavirus is critical for early diagnosis and control of severe acute respiratory syndrome. This study describes a method for the detection of SARS associated coronavirus gene by the combination of functionalized nanoparticles and PCR-based assay. In this method, the target cDNA of SARS associated coronavirus was firstly captured and enriched from the mixture of target cDNA and non-target cDNA by the use of the functionalized silica coated superparamagnetic nanoparticles. Additionally, the enriched target cDNA were amplified through a general symmetry PCR and then was selectively isolated from the double strands PCR products by applying the silica coated superparamagnetic nanoparticles again. Finally, we detected the amplified target cDNA by employing the functionalized silica coated fluorescent nanoparticles as signaling probes with a sandwich hybridization format. The results show that the target cDNA can be assayed successfully with a detection limit of 2.0 x 10(3) copies and the nonspecific amplification could be inhibited. In addition, the detection procedure is rapid and can be completed in less than 6 h. Our results suggest that this approach will provide promising prospects for other pathogens detection.     

One-step homogeneous protein detection based on aptamer probe and fluorescence cross-correlation spectroscopy

A new protein assay based on fluorescence cross-correlation spectroscopy (FCCS) and aptamer probe is developed. In this assay, two spectrally distinct fluorophores labeled aptamer probes are used to recognize and detect thrombin through a sandwich reaction. The sandwich complexes are diffused through a confocal detection volume. The cross-correlation signals can be observed only at the presence of the aptamer probes-protein sandwich complexes. Thrombin is selected as a target to validate the assay. The whole detection process can be completed within an hour with low-nanomolar sensitivity and high specificity. The novel aptamer-based FCCS detection offers a simple, rapid and sensitive method for protein analysis in a homogeneous format.     

Lab-on-a-bubble surface enhanced Raman indirect immunoassay for cholera

We describe a novel sandwich assay based on surface enhanced Raman scattering (SERS) comprised of buoyant silica microspheres coated with antibodies against the β subunit of the cholera toxin (CT), and gold nanoparticles tagged with a Raman reporter, shelled with silica and coated with antibodies against the β subunit of the CT. Together these components couple to form a sandwich which, after incubation, floats on the surface of the sample. The buoyant silica microparticle/nanoparticle reporter combination has been coined a lab on a bubble (LoB). LoB materials may provide a platform for rapid detection of antigen in solution and offers advantages over lateral flow or magnetic pull-down assays. The Raman reporter provides a unique and intense signal to indicate a positive analysis. Our limit of detection for the β subunit of the CT in a buffer based system is 1100 ng.     

Fast colorimetric detection of copper ions using L-cysteine functionalized gold nanoparticles

This communication reports an efficient visual detection method of Cu2+ by L-cysteine functionalized gold nanoparticles in aqueous solution. Upon exposure to Cu2+, the gold nanoparticle solution changed from red to blue, in response to surface plasmon absorption of dispersed and aggregated nanoparticles. This colorimetric sensor allows a rapid quantitative assay of Cu2+ down to the concentration range of 10(-5) M. Recognition of Cu2+ and formation of the aggregates are proposed to occur via a 2 : 1 sandwich complex between L-cysteine and Cu2+.     

Fluorescent ferritin nanoparticles and application to the aptamer sensor

We synthesized fluorescent ferritin nanoparticles (FFNPs) through bacterial expression of the hybrid gene consisting of human ferritin heavy chain (hFTN-H), spacer (glycine-rich peptide), and enhanced green (or red) fluorescent protein [eGFP (or DsRed)] genes. The self-assembly activity of hFTN-H that leads to the formation of nanoparticles (12 nm in diameter), the conformational flexibility of the C-terminus of hFTN-H, and the glycine-rich spacer enabled eGFPs (or DsReds) to be well displayed on the surface of each ferritin nanoparticle, resulting in the construction of green (or red) FFNPs [gFFNPs (or rFFNPs)]. As compared to eGFP (or DsRed) alone, it is notable that the developed FFNPs showed significantly amplified fluorescence intensity and also enhanced stability. DNA aptamers were chemically conjugated to gFFNP via each eGFP's cysteine residue that was newly introduced through site-directed mutagenesis (Ser175Cys). The DNA-aptamer-conjugated gFFNPs were used as a fluorescent reporter probe in the aptamer-based "sandwich" assay of a cancer marker [i.e., platelet-derived growth factor B-chain homodimer (PDGF-BB)] in phosphate-buffered saline buffer or diluted human serum. This is a simple two-step assay without any additional steps for signal amplification, showing that compared to the same aptamer-based assays using eGFP alone or Cy3, the detection signals, affinity of the reporter probe to the cancer marker, and assay sensitivity were significantly enhanced; i.e., the limit of detection was lowered to the 100 fM level. Although the PDGF-BB assay is reported here as a proof-of-concept, the developed FFNPs can be applied in general to any aptamer-based sandwich assays.     

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.     

Optical Detection of Human Papillomavirus Type 16 and Type 18 by Sequence Sandwich Hybridization With Oligonucleotide-Functionalized Au Nanoparticles

The importance of detecting and subtyping human papillomaviruses (HPVs) in clinical and epidemiological studies has been well addressed. In detecting the most common types of HPV, type 16 (HPV-16) and type 18 (HPV-18), in the cervical mucous of patients in a simple and rapid manner, the assay of a label-free colorimetric DNA sensing method based on sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles (AuNPs) was fabricated in this study. Specific oligonucleotide probes were designed for the sequence detection within the L1 gene of HPV-16 and HPV-18, and the probes were capped onto AuNPs, as AuNP probes. The target HPV sequences in clinical specimens were obtained by an asymmetric polymerase chain reaction (PCR) with universal primers, which can amplify the target sequences from several HPV serotypes, including HPV-16 and HPV-18. The DNA sandwich hybridization between the target sequences and the specific AuNP probes was performed at a temperature closer to the theoretical melting temperature of the DNA hybridization. Next, the procedure of increasing salt concentration and cooling the hybridizing solution was immediately utilized to discriminate the target sequences of HPV-16 or HPV-18. If the target sequences were not complementary to sequences of AuNP probes, the AuNPs would aggregate because no duplex DNA formation occurred such that the color of the reaction solution changed from red to purple. If the AuNP probes were a perfect match to the target sequences and a full DNA sandwich hybridization occurred, the reaction solution maintained its red color. A total of 70 mucous specimens from patients with cervical intraepithelial neoplasia were tested by the AuNP probes sandwich hybridization.      

Homogeneous detection of nucleic acids based upon the light scattering properties of silver-coated nanoparticle probes

Herein we report the development of a simple, rapid, homogeneous, and sensitive detection system for DNA based on the scattering properties of silver-amplified gold nanoparticle probes. The assay uses DNA-functionalized magnetic particle probes that act as scavengers for target DNA, which can be collected via a magnetic field. Once the DNA targets are isolated from the initial sample, they are sandwiched via hybridization by a second set of probes. The latter probes are 13-nm gold nanoparticles modified with a different target complementary DNA. Excess probes are removed through repetitive washing steps. The gold particles are dispersed in solution by dehybridization, corresponding to an assumed 1:1 ratio with the target DNA. Electroless deposition of silver on the surface of the gold probes results in particle growth, which increases their scattering efficiency with time. The scattering efficiency and the extinction signatures of the particle sizes are monitored as a function of time and correlated with target concentration. The limit of detection for this novel assay was determined to be 10 fM.     

Anionic solid lipid nanoparticles supported on protamine/DNA complexes

The objective of this study was to design novel anionic ternary nanoparticles for gene delivery. These ternary nanoparticles were equipped with protamine/DNA binary complexes (150-200?nm) as the support, and the anionic formation was achieved by absorption of anionic solid lipid nanoparticles (≤20?nm) onto the surface of the binary complexes. The small solid lipid nanoparticles (SLNs) were prepared by a modified film dispersion-ultrasonication method, and adsorption of the anionic SLNs onto the binary complexes was typically carried out in water via electrostatic interaction. The formulated ternary nanoparticles were found to be relatively uniform in size (257.7 ± 10.6?nm) with a 'bumpy' surface, and the surface charge inversion from 19.28 ± 1.14?mV to -17.16 ± 1.92?mV could be considered as evidence of the formation of the ternary nanoparticles. The fluorescence intensity measurements from three batches of the ternary nanoparticles gave a mean adsorption efficiency of 96.75 ± 1.13%. Circular dichroism spectra analysis showed that the protamine/DNA complexes had been coated by small SLNs, and that the anionic ternary nanoparticles formed did not disturb the construction of the binary complexes. SYBR Green I analysis suggested that the ternary nanoparticles could protect the DNA from nuclease degradation, and cell viability assay results showed that they exhibit lower cytotoxicity to A549 cells compared with the binary complexes and lipofectamine. The transfection efficiency of the ternary nanoparticles was better than that of naked DNA and the binary complexes, and almost equal to that of lipofectamine/DNA complexes, as revealed by inversion fluorescence microscope observation. These results indicated that the anionic ternary nanoparticles could facilitate gene transfer in cultured cells, and might alleviate the drawbacks of the conventional cationic vector/DNA complexes for gene delivery in vivo.     

Paper-based tuberculosis diagnostic devices with colorimetric gold nanoparticles

Abstract

A colorimetric sensing strategy employing gold nanoparticles and a paper assay platform has been developed for tuberculosis diagnosis. Unmodified gold nanoparticles and single-stranded detection oligonucleotides are used to achieve rapid diagnosis without complicated and time-consuming thiolated or other surface-modified probe preparation processes. To eliminate the use of sophisticated equipment for data analysis, the color variance for multiple detection results was simultaneously collected and concentrated on cellulose paper with the data readout transmitted for cloud computing via a smartphone. The results show that the 2.6 nM tuberculosis mycobacterium target sequences extracted from patients can easily be detected, and the turnaround time after the human DNA is extracted from clinical samples was approximately 1 h.     

Ultrasensitive DNA detection using oligonucleotide-silver nanoparticle conjugates

Oligonucleotide-gold nanoparticle (OGN) conjugates are powerful tools for the detection of target DNA sequences due to the unique properties conferred upon the oligonucleotide by the nanoparticle. Practically all the research and applications of these conjugates have used gold nanoparticles to the exclusion of other noble metal nanoparticles. Here we report the synthesis of oligonucleotide-silver nanoparticle (OSN) conjugates and demonstrate their use in a sandwich assay format. The OSN conjugates have practically identical properties to their gold analogues and due to their vastly greater extinction coefficient both visual and absorption analyses can occur at much lower concentrations. This is the first report of OSN conjugates being successfully used for target DNA detection and offers improved sensitivity which is of interest to a range of scientists.     

包装印刷品条码质量检测方法

目的为保证商品条码在物流系统中的快速识别和信息传递,研究条码的质量检测方法。方法首先分出条码区域,考虑到条码的特殊属性,需要满足其可识读功能,设计针对EAN-13商品条码的印制质量检测方法,包括可识读检测和印刷缺陷检测。根据条码检测的国家标准,条码可识读检测部分,采用扫描反射率曲线分析法和条码质量分级法对条码的可识读性进行判定。条码缺陷检测部分经过条码校正、条码与字符的分割和条码大小的归一化等处理后,选定基于垂直投影的缺陷检测算法对条码的脱墨和污点缺陷进行检测。结果条码识读程序对合格品和缺陷品的识读准确率都为100%,条码缺陷检测算法程序的平均检测耗时为93.35 ms,检测准确率为94%。结论条码质量检测系统具有较高的检测准确率,并且能够很好地满足机器视觉缺陷检测速度的要求。     

Nonenzymatic detection of bacterial genomic DNA using the bio bar code assay

The detection of bacterial genomic DNA through a nonenzymatic nanomaterials-based amplification method, the bio bar code assay, is reported. The assay utilizes oligonucleotide-functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double-stranded DNA. These blockers bind to specific regions of the target DNA upon cooling and prevent the duplex DNA from rehybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide-functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (bar codes) act as amplification surrogates. The bar codes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 fM, and this is the first demonstration of a bar code-type assay for the detection of double-stranded, genomic DNA.     

Enhanced Sensitivity in Nanopore Sensing of Cancer Biomarkers in Human Blood via Click Chemistry

Cancer biomarkers are expected to be indicative of the occurrence of certain cancer diseases before the tumors form and metastasize. However, many biomarkers can only be acquired in extremely low concentrations, which are often beyond the limit of detection (LOD) of current instruments and technologies. A practical strategy for nanopore sensing of cancer biomarkers in raw human blood down to the femtomolar level is developed here. This strategy first converts the detection of cancer biomarkers to the quantification of copper ions by conducting a sandwich assay involving copper oxide nanoparticles. The released Cu²⁺ is then taken to catalyze the “click” reaction which ligates a host–guest modified DNA probe. Finally, this DNA probe is subjected to single‐channel recordings to afford the translocation events that can be used to derive the concentrations of the original biomarkers. Due to the amplification effects of nanoparticle loadings and the “click” reaction, the LOD of this strategy can be as low as the subfemtomolar level. Further, the acid treatment step could effectively eliminate the interferences from plasma proteins in raw human blood and make the strategy highly suitable for the detection of cancer biomarkers in clinical samples.     

Portable and quantitative detection of protein biomarkers and small molecular toxins using antibodies and ubiquitous personal glucose meters

Developing portable and low-cost methods for quantitative detection of large protein biomarkers and small molecular toxins can play a significant role in controlling and preventing diseases or toxins outbreaks. Despite years of research, most current methods still require laboratory-based or customized devices that are not widely available to the general public for quantitative analysis. We have previously demonstrated the use of personal glucose meters (PGMs) and functional DNAs for the detection of many nonglucose targets. However, the range of targets detectable by functional DNAs is limited at the current stage. To expand the range of targets that can be detected by PGMs, we report here the use of antibodies in combination with sandwich and competitive assays for quantitative detection of protein biomarkers (PSA, with a detection limit of 0.4 ng/mL) and small molecular toxins (Ochratoxin A, with a detection limit of 6.8 ng/mL), respectively. In both assay methods, with invertase conjugates as the link, quantitative detection is achieved via the dependence between the concentrations of the targets in the sample and the glucose measured by PGMs. Given the wide availability of antibodies for numerous targets, the methods demonstrated here can expand the range of target detection by PGMs significantly.     

Immunoassay readout method using extrinsic Raman labels adsorbed on immunogold colloids

An immunoassay readout method based on surface-enhanced Raman scattering (SERS) is described. The method exploits the SERS-derived signal from reporter molecules that are coimmobilized with biospecific species on gold colloids. This concept is demonstrated in a dualanalyte sandwich assay, in which two different antibodies covalently bound to a solid substrate specifically capture two different antigens from an aqueous sample. The captured antigens in turn bind selectively to their corresponding detection antibodies. The detection antibodies are conjugated with gold colloids that are labeled with different Raman reporter molecules, which serve as extrinsic labels for each type of antibody. The presence of a specific antigen is established by the characteristic SERS spectrum of the reporter molecule. A near-infrared diode laser was used to excite efficiently the SERS signal while minimizing fluorescence interference. We show that, by using different labels with little spectral overlap, two different antigenic species can be detected simultaneously. The potential of this concept to function as a readout strategy for multiple analytes is briefly discussed.     

Detection of antigens using a protein-DNA chimera developed by enzymatic covalent bonding with phiX gene A*

The chemical reactions used to make antibody-DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of φX174 gene A* protein and Z(mab25) (A*-Zmab). The φX174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab25) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin γ1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-γ (IFN-γ) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.