Lateral flow dipstick test for genotyping of 15 beta-globin gene (HBB) mutations with naked-eye detection

For definitive diagnosis of thalassemia carriers and patients, as well as for prenatal diagnosis, genotype analysis is of fundamental importance. We report a dry-reagent, lateral flow dipstick test that enables visual genotyping (detection by naked eye) of 15 mutations common in Mediterranean populations in the beta-globin gene (HBB). The method comprises 3 simple steps: (i) PCR amplification of a single 1896 bp segment of the beta globin gene flanking all 15 mutations; (ii) a multiplex (10-plex and/or 30-plex) primer extension reaction of the unpurified amplification product using allele-specific primers. Biotin is incorporated in the extended product; (iii) a dry-reagent multi-allele (10-plex) dipstick assay for visual detection of the primer extension reaction products within minutes. The total time required for PCR, primer extension reaction and the dipstick assay is ∼2 h. The method was evaluated by genotyping 45 DNA samples of known genotypes and 54 blind samples. The results were fully concordant with reference methods. The method is simple, rapid, and cost-effective. Detection by the dipstick assay does not require specialized instrumentation or highly qualified personnel. The proposed method could be a particularly useful tool in laboratories with limited resources and a basis for point-of-care diagnostics especially in combination with PCR amplification from whole blood.     

Cholinesterase-based dipstick assay for the detection of organophosphate and carbamate pesticides

A cholinesterase (ChE)-based dipstick-type assay for the class-specific detection of organophosphate (OP) and carbamate (CM) pesticides was developed. The principle of the assay is based on inhibition of the activity of a ChE by these two families of pesticides, which is dependent on the concentration of pesticides. The proposed assay system is composed of a test strip with an acetylcholinesterase (AChE)-coated membrane and an enzyme substrate solution. The assay protocol involves incubation of the enzyme-coated strip in the pesticide-containing sample solution followed by incubation of the sample-treated strip in a chromogenic enzyme substrate solution. The color intensity is estimated by the naked eye or a reflectometer. Of the membranes tested as the enzyme support, Hybond N⁺ was the most suitable. Among the compounds tested as the enzyme substrate, indophenyl acetate was the best. The detectable concentration range of the dipstick assay for the OP and CM pesticides was 10⁻⁶-10² and 10⁻⁶-10⁰ μg mL⁻¹, respectively. The sensitivity of the dipstick assay to the oxidized form of parathion (paraoxon) was higher than to parathion. The strip showed a large matrix effect with pesticide-spiked lettuce samples, whereas it showed a small matrix effect with pesticide-spiked rice samples.     

Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals

A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin–BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73–109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 μg kg⁻¹, respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 μg kg⁻¹, respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC–MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.     

Ultrasonic Synthesis of Gold Nanoparticles and Its Use in Immunochromatographic Assay for Detection of Kanamycin

An ultrasonic method to synthesize gold nanoparticles with uniform size was reported. Effects of ethanol concentration, solution pH, ultrasonic irradiation power, and time on the formation of gold nanoparticles were investigated. The obtained gold nanoparticles were characterized by ultraviolet-visible (UV-vis) absorption spectra and scanning electron microscope (SEM). The method performed in ethanol solution under ultrasonic irradiation is friendly to the environment. Furthermore, a nanogold-labeled probe was used to develop an immunochromatographic method for detection of kanamycin.     

A simple double-bead sandwich assay for protein detection in serum using UV-vis spectroscopy

In this study a double-bead sandwich assay, employing magnetic nanoparticles and gold nanoparticles is proposed. The magnetic nanoparticles allow specific capturing of the analyte in biological samples, while the optical properties of the gold nanoparticles provide the signal transduction. We demonstrated that a major improvement in the assay sensitivity was obtained by selecting an optimal gold nanoparticle size (60 nm). A detection limit of 5-8 ng/mL, a sensitivity of 0.6-0.8 (pg/mL)⁻¹ and a dynamic range of 3 orders of magnitude were achieved without any further amplification using the detection of prostate specific antigen in serum as a model system. The proposed assay has the ability to be easily implemented within a microfluidic device for point-of-care applications whereby the readout can be executed by a fast and cheap optical measurement.     

A naked-eye based strategy for semiquantitative immunochromatographic assay

It is critical to develop a cost-effective quantitative/semiquantitative assay for rapid diagnosis and on-site detection of toxic or harmful substances. Here, a naked-eye based semiquantitative immunochromatographic strip (NSI-strip) was developed, on which three test lines (TLs, TL-I, TL-II and TL-III) were dispensed on a nitrocellulose membrane to form the test zone. Similar as the traditional strip assay for small molecule, the NSI-strip assay was also based on the competitive theory, difference was that the analyte competed three times with the capture reagent for the limited number of antibody binding sites. After the assay, the number of TLs developed in the test zone was inversely proportional to the analyte concentration, thus analyte content levels could be determined by observing the appeared number of TLs. Taking aflatoxin B₁ as the model analyte, visual detection limit of the NSI-strip was 0.06 ng mL⁻¹ and threshold concentrations for TL-I–III were 0.125, 0.5, and 2.0 ng mL⁻¹, respectively. Therefore, according to the appeared number of TLs, the following concentration ranges would be detectable by visual examination: 0–0.06 ng mL⁻¹ (negative samples), and 0.06–0.125 ng mL⁻¹, 0.125–0.5 ng mL⁻¹, 0.5–2.0 ng mL⁻¹ and >2.0 ng mL⁻¹ (positive samples). That was to say, compared to traditional strips the NSI-strip could offer more parameter information of the target analyte content. In this way, the NSI-strip improved the qualitative presence/absence detection of traditional strips by measuring the content (range) of target analytes semiquantitatively.     

Gold immunochromatographic strips for enhanced detection of Avian influenza and Newcastle disease viruses

A new technique that uses gold immunochromatographic strips enhances the detection sensitivity by inducing the clustering of additional gold nanoparticles (AuNPs) around the immunogold particles immobilized on nitrocellulose strips. The additional AuNPs provide an intense signal that can be detected by the naked eye. The AuNPs were synthesized and conjugated to monoclonal antibodies using self-assembly. Other antibodies were immobilized in a defined detection zone on the nitrocellulose membrane. The detection principle is based on a “sandwich” immunoreaction, where gold-labeled antibodies serve as signal vehicles. To improve the sensitivity of the strips, we use a mixture of 1% HAuCl₄ and 10 mmol L⁻¹ NH₂OH·HCl to “enlarge” the gold nanoparticles. The detecting limits of Avian influenza virus (AIV) and Newcastle disease virus (NDV) are significantly increased. Compared with commercial test strips, this method is 100-fold more sensitive. This method is easy to perform and can be carried out on-site in test laboratories.     

A one-step immunochromatographic assay for detecting ginsenosides Rb1 and Rg1

An immunochromatographic strip test has been developed for detecting ginsenosides Rb1 (G-Rb1) and Rg1 (G-Rg1). This qualitative assay system is useful as a rapid screening method for detecting G-Rb1 and G-Rg1 in plants and plant preparations. Our assay is a competitive immunoassay that uses anti-G-Rb1 and anti-G-Rg1 monoclonal antibodies (MAbs) and a detection reagent that contains colloidal gold particles coated with anti-G-Rb1 and anti-G-Rg1 MAbs. Detection limits are 2 g mL^–1 for both G-Rb1 and G-Rg1.     

A novel enhancement assay for immunochromatographic test strips using gold nanoparticles

The immunochromatographic assay is a well-known and convenient diagnostic system. In this report, the development of a novel enhancement assay for the test strips is described. Additionally, this highly sensitive immunochromatographic assay was applied to detect human chorionic gonadotropin hormone (HCG) as the model case. The primary antibody-conjugated gold nanoparticles were used as the enhancer of the standard method. The primary antibodies were immobilized within a defined detection zone (test line) on the diagnostic nitrocellulose membrane. The secondary antibodies were conjugated with colloidal gold nanoparticles. In combination with an effective sample pretreatment, the gold-conjugated antibodies and the primary antibodies formed a sandwich complex with the target protein. Within the test line, the sandwich complex was immobilized, and furthermore, concentrated by the enhancer resulting in a localized surface plasmon resonance (LSPR) phenomenon and a distinct red color on the test line. The intensity of color of the red test line (signal intensity), which correlated directly with the concentration of the target protein in the standard or spiked samples, was assessed visually and by computer image analysis using a three-determination analysis. Under optimum conditions, the limit of detection (LOD) for HCG assay was 1 pg/mL. When using human serum, 10 pg/mL of HCG could be detected. We have also spiked total prostate-specific antigen (TPSA) in female serum. The LOD for TPSA was determined as 0.2 ng/mL. With this method, the quantitative determination of the target protein could be completed in less than 15 min. Our novel immunochromatographic strips using the enhancing method based on LSPR of gold nanoparticles are useful as a rapid and simple screening method for the detection of important analytes for medical applications, environmental monitoring, food control, and biosecurity.      

Detection of flavonoids and assay for their antioxidant activity based on enlargement of gold nanoparticles

We report our findings that natural flavonoids such as quercetin, daizeol and puerarin can act as reductants for the enlargement of gold nanoparticles (Au-NPs). Consequently, the UV–vis spectra of a solution containing Au-NPs will be gradually changed, and the molecules of the natural herbs can be detected by making use of changes in the UV–visible spectra. Furthermore, we have prepared a self-assembled monolayer modified electrode by modifying cysteamine on a gold substrate electrode, which is further modified by some Au-NP seeds. When the modified electrode is immersed in a solution containing flavonoids and tetrachloroauric acid as a gold source for the growth of the Au-NP seeds, with the increase of the concentration of flavonoids, the Au-NP seeds on the surface of the modified electrode can be enlarged to varying degrees. As a result, the peak currents in the corresponding cyclic voltammograms are inversely decreased, and simultaneously the peak separation is increased. Therefore, an electrochemical method to detect flavonoids is also proposed. Compared with the optical detection method, the electrochemical method has an extraordinarily lower detection limit and a significantly extended detection range. Moreover, the optical and electrochemical experimental results can be also used to assay and compare the relative antioxidant activities of the flavonoids. Figure Enlargement of Au nanoparticles by flavonoids at cysteamine modified electrode     

A new quality control method for lateral flow assay

We proposed a lateral flow assay (LFA) based on internal quality control microspheres to realize the accurate diagnosis of HbA1c in human body. This method can improve the precision and accuracy of HbA1c detection.     

Optimization of a lateral flow immunoassay for the ultrasensitive detection of aflatoxin M1 in milk

A high sensitive immunoassay-based lateral flow device for semi-quantitatively determine aflatoxin M₁ (AFM₁) in milk was developed. Investigation and optimization of the competitor design and of the gold-labelling strategy allowed the attainment of the ultra-sensitive assessment of AFM₁ contamination at nanograms per litre level (LOD 20 ng L⁻¹, IC₅₀ 99 ng L⁻¹), as requested by European regulations. A one order of magnitude detectability enhancement in comparison to previously reported gold colloid immunochromatographic assays for this toxin was obtained.     

A gold nanoparticle labeling strategy for the sensitive kinetic assay of the carbamate-acetylcholinesterase interaction by surface plasmon resonance

The article presents a novel strategy for a sensitive investigation of the interaction between acetylcholinesterase (AChE) and its small molecular carbamate inhibitors. Two carbamate inhibitors with different ether linkages and the terminal lipoate were synthesized and labeled with gold nanoparticles (AuNPs). With the signal amplification of AuNPs, the specific interactions between the AuNPs labeled carbamate inhibitors (ALC1 and ALC2) and the immobilized AChE on sensor chip surface were readily examined. The detection sensitivities of ALC1 and ALC2 were 176 and 121 m°/nM, respectively, with the detection limits of 7.0 and 12 pM at a signal-to-noise ratio of 3. The association/dissociation constants for the binding interaction between carbamate inhibitors and AChE were reported for the first time. The affinity constants were estimated to be 3.13 × 10⁶ and 6.39 × 10⁵ M⁻¹ for ALC1 and ALC2 respectively. This AuNPs labeling strategy is versatile and may be applicable for the direct or competitive SPR kinetic assay of the interaction between small molecule inhibitors and their target proteins with a high sensitivity.     

Staining-free gel electrophoresis-based multiplex enzyme assay using DNA and peptide dual-functionalized gold nanoparticles

We report a simple staining-free gel electrophoresis method to simultaneously probe protease and nuclease. Utilizing gold nanoparticles (Au-NPs) dual-functionalized with DNA and peptide, the presence and concentration of nuclease and protease are determined concurrently from the relative position and intensity of the bands in the staining-free gel electrophoresis. The use of Au-NPs eliminates the need for staining processes and enables naked eye detection, while a mononucleotide-mediated approach facilitates the synthesis of DNA/peptide conjugated Au-NPs and simplifies the operation procedures. Multiplex detection and quantification of DNase I and trypsin are successfully demonstrated.     

A selective,sensitive and label-free visual assay of fructose using anti-aggregation of gold nanoparticles as a colorimetric probe

A new convenient colorimetric sensor for fructose based on anti-aggregation of citrate-capped gold nanoparticles(Au NPs) is presented. 4-Mercaptophenylboronic acid(MPBA) induces the aggregation of Au NPs, leading to a color change from red to blue. Fructose as a potent competitor has strong affinity for MPBA and a borate ester is formed between MPBA and fructose. There is an obvious color change from blue to red with increasing the concentration of fructose. The anti-aggregation effect of fructose on Au NPs was seen by the naked eye and monitored by UV–vis spectra. Our results showed that the absorbance ratio(A_(519)/A_(640)) was linear with fructose concentration in the range of 0.032–0.96 μmol/L(R~2= 0.996), with a low detection limit of 0.01 μmol/L(S/N = 3). Notably, a highly selective recognition of fructose was shown against other monosaccharide and disaccharide(glucose, mannose, galactose,lactose and saccharose). With anti-aggregation assays higher selectivity is achievable. The results of this work provide a rapid method for evaluating the quantitative analysis of fructose in human plasma at physiologically meaningful concentrations and at neutral pH. The proposed procedure can be used as an efficient method for the precise and accurate determination of fructose.     

上转换发光免疫层析试纸条快速定量检测己烯雌酚

采用溶剂热法合成上转换纳米颗粒(UCNPs)-NaYF4∶ Yb,Er,进行表面功能化修饰,将其与己烯雌酚(DES)单克隆抗体偶联,制备荧光探针,以牛血清白蛋白-己烯雌酚(BSA-DES)偶联物和羊抗鼠二抗分别喷涂硝酸纤维膜,形成试纸条检测线(T线)和质控线(C线),建立了上转换发光免疫层析试纸条快速定量检测DES的方法.实验结果表明,此试纸条定量检测DES线性范围为25～ 10000 ng/mL(y=0.43927x-0.57647,R2=0.996),检出限为20.84 ng/mL,单个样品检测时间为15 min,批内和批间变异系数均小于10％,特异性识别能力强,在37℃存放下7天检测值RSD的平均值约为15％.加标回收实验显示平均回收率为90.1％～115.2％,相对标准偏差小于5％,与高效液相色谱法有较好的一致性.     

A sensitive and semi-quantitative method for determination of multi-drug residues in animal body fluids using multiplex dipstick immunoassay

The objective of this research was to develop a multiplex dipstick immunoassay method for the simultaneous determination of multi-veterinary drug residues, such as β-agonists, sulfonamides, and tetracyclines in milk, urine, and serum. The multiplex dipstick assay format was based on an indirect competitive approach: Three test lines (different antigens) and one control line (goat anti-mouse IgG) were located on the strip membrane. Labeled antibodies were freeze-dried in microwells. Samples did not require pretreatment and could be directly analyzed within 10 min. Threshold levels in different sample matrices were visually estimated at 0.3–0.45 ng mL⁻¹ for clenbuterol; 3–4 ng mL⁻¹ for sulfadiazine; and 4.5–6 ng mL⁻¹ for tetracycline, respectively. The linear relationship between the concentrations of veterinary drug residues and the Au nanoparticles plasmon absorbance allowed quantitative determination of these veterinary drug residues. The recoveries of clenbuterol, sulfadiazine and tetracycline in spiked samples ranged from 78.4% to 112.6%, and the relative standard deviations were below 11.2%. Analysis of animal samples suggested that the proposed multiplex dipstick assay method was consistent with the LC-MS/MS method. The percentage of false results was less than or equal to 5%. Thus, the proposed multiplex dipstick assay is inexpensive, easy-to-use, and suitable for the purposes of rapid and comprehensive screening of 3 families of β-agonists, sulfonamides and tetracyclines including 26 drugs in animal body fluids.     

A sensitive immunosorbent bio-barcode assay combining PCR with icELISA for detection of gonyautoxin 2/3

In the current study, we developed a nanosphere bio-barcode technology to detect trace gonyautoxin 2/3 (GTX 2/3). GTX 2/3-glucose oxidase (GOX) conjugates were first prepared as the coating antigen in a periodate reaction. Subsequently, gold nanoparticles (NP) dual-labeled with anti-GTX 2/3 monoclonal antibodies (Mab) and DNA oligonucleotides were synthesized via a one-step preparation method. Combining PCR with indirect competitive ELISA (icELISA), a novel immunosorbent bio-barcode assay was established utilizing the Mab-NP-dsDNA complex to convert enzymatic signals to DNA signals. Importantly, the limit of detection of the method was lower than 0.74 μg mL⁻¹. Thus, the immunosorbent bio-barcode assay is a rapid and high-throughput screening tool to detect GTX 2/3 in aquatic products.     

Multi-residue detection of pesticides using a sensitive immunochip assay based on nanogold enhancement

This paper describes the development of a new multiplex immunoassay for simultaneous detection of seven pesticides (triazophos, methyl-parathion, fenpropathrin, carbofuran, thiacloprid, chlorothalonil, and carbendazim). Sixteen pairs of pesticide antibodies and antigens were screened for reactivity and cross-reaction. A microarray chip consisting of seven antigens immobilized on a nitrocellulose membrane was then constructed. Nanogold was employed for labeling and signal amplification to obtain a sensitive colorimetric immunoassay. The direct and indirect detection formats were further compared using primary antibody-gold and secondary antibody-gold conjugates as tracers. An integrated 7-plex immunochip assay based on the indirect model was established and optimized. The detection limits for the pesticides were 0.02–6.45 ng mL⁻¹, which meets detection requirements for pesticide residues. Naked-eye assessment showed the visual detection limits of the assay ranged from 1 to 100 ng mL⁻¹. Spiked recovery results demonstrated that the immunochip assay had potential for multi-analysis of pesticide residues in vegetables and fruits. The proposed microarray methodology is a flexible and versatile tool, which can be applied to other competitive multiplex immunoassays for small molecular compounds.