Study on Colloidal Gold Immunochromatography Assay for Rapid Detection of Spectinomycin

[Objectives] This study was conducted to establish a rapid detection method for spectinomycin in pork,chicken,fish,shrimp flesh and water.[Methods]A test strip for rapid detection of spectinomycin in milk was developed by colloidal gold immunochromatography assay. [Results]The test strip had a detection limit of 50 μg/kg to milk with a detection time of 15 min,and the false positive rate and false negative rate were both 0. [Conclusions]The method is accurate,simple,reliable and convenient,and is suitable for rapid on-site spectinomycin detection.

Colloidal Gold Immunochromatographic Assay for Rapid On-Site Detection of Tetracycline in Seawater

Recently increasing concerns from the scientists and public have been paid for seawater pollution due to tetracycline(TC)overuse in maricultural area.However,there are few methods or instruments that can be used for specific and rapid detection of this antibiotic in seawater.In this study,the colloidal gold immunochromatographic assay(CG-ICA)was used to achieve this goal.A commercialized monoclonal antibody against TC(anti-TC mAb)was selected because of its higher sensitivity(half-maximal inhibitory concentration of 2.38μgL^(-1)).The prepared CG particles(average diameter of 20 nm)were used to label anti-TC mAb at pH 8.0.The conjugate pad was formed by spraying the CG-labeled anti-TC mAb on a glass fibre membrane followed by proper dryness.The test pad was made by immobilizing artificial antigen and anti-mouse mAb in the test line and the control line,respectively,in a nitrocellulose membrane.The test strip,assembled with sample pad,conjugate pad,test pad and absorbent pad,could be used to detect TC during seawater sample flowing through these components in turn.The results could be observed by the naked eye in 10min.The visible limit of detection(vLOD)was 20μgL^(-1) for TC in seawater.The CG-ICA test results were in good agreement with those of liquid chromatography-tandem mass spectrometry(LC-MS/MS).The assay also showed that,oxytetracycline(OTC)and chlortetracycline(CTC),as the structural analogues of TC,did not interfere with TC determination.Furthermore,the TC concentration given by test strip could not be affected by the fluctuation of temperature(10℃–30℃),pH(7–9)and salinity(0–40)of seawater.Therefore,CG-ICA is a suitable tool for rapid,on-site,and semi-quantitative detection of TC in seawater.

Effects of 105 traditional Chinese medicines on the detection ofβ-agonists in medicine extracts and swine urine based on colloidal gold immunochromatographic assay

Colloidal gold immunochromatographic assay(CGIA)is commonly used for the on-site detection ofβ-agonists that are sometimes used illegally as feed additives in swine diets.However,few studies have evaluated the causes of false-positive results that sometimes occur when applying CGIA in agricultural settings.In this study,we investigated if this false-positive phenomenon is related to the addition of certain traditional Chinese medicines(TCMs)to swine feed.We established and verified an extraction method for TCMs,and then applied CGIA to detectβ-agonists in the extracts of 105 TCMs and in the urine of swine dosed with TCMs,respectively.Liquid chromatography-tandem mass spectrometry was used to validate the results of the urine samples tested positive forβ-agonists using CGIA.The results were also verified using TCMs and colloidal gold test strips produced by different manufacturers.The extracts of Citri Reticulatae Pericarpium Viride,Citri Reticulatae Pericarpium,Magnoliae Officinalis Cortex,Chaenomelis Fructus,and Rhodiolae Crenulatae Radix Et Rhizoma were tested positive forβ-agonists.Meanwhile,the addition of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium to swine feed resulted in false-positive results forβ-agonists in swine urine.The results provide a new way to explain false-positive CGIA results and provide valuable information for livestock feeding programs.

Lectin Conjugated Gold Nanoparticle-based Colorimetric Assay for Studying the Interactions of Antibiotic with Living Cell

The interactions of antibiotic with living cells were studied by lectin conjugated gold nanoparticles（GNPs） based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotic, tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay. In the presence of tunicamycin, the dose- and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy（CLSM） and classic flow cytometry（FCM） assay, and satisfactory results were obtained.

Gold Nanoparticle-based Colorimetric Assay for Determination of Lead(Ⅱ) in Aqueous Media

The authors presented a simple colorimetric assay for the detection of toxic heavy metal lead（Ⅱ） ion（Pb2＋） Pentapeptide, cysteine-alanine-leacine-asparagine-asparagine（CALNN）, functionalized gold nanoparticles（GNPs） were aggregated in the presence of the divalent metal ion in solution by an ion-templated chelation process, which caused an easily measurable change in the absorption spectrum of the particles. Typically, mono-dispersing GNPs exhibit an absorption band at 522 nm, corresponding to a red color solution, while aggregated GNPs have it at longer wavelengths, corresponding to a purple or blue color solution. The chelation/aggregation process is reversible via the addition of a strong metal ion chelator such as EDTA. Highly selective and sensitive detection of Pb^2＋ in aqueous solution is thus provided. A detection limit of 0.1 μmol/L of Pb^2＋ was demonstrated.

Rapid one-step enzyme immunoassay and lateral flow immunochromatographic assay for colistin in animal feed and food

Background:Colistin(polymyxin E)is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention,treatment,and growth promotion.However,the wide use of colistin in animal feed may accelerate the spread of colistin-resistance gene MCR-1 from animal production to human beings,and its residue in animal-origin food may also pose serious health hazards to humans.Thus,it is necessary to develop corresponding analytical methods to monitor the addition of colistin in animal feed and the colistin residue in animal-origin food.Results:A one-step enzyme-linked immunosorbent assay(ELISA)and a lateral flow immunochromatographic assay(LFIA)for colistin were developed based on a newly developed monoclonal antibody.The ELISA showed a 50%inhibition value(IC50)of 9.7 ng/m L with assay time less than 60 min,while the LFIA had a strip reader-based detection limit of 0.87 ng/m L in phosphate buffer with assay time less than 15 min.For reducing the non-specific adsorption of colistin onto sample vial,the components of sample extraction solution were optimized and proved to greatly improve the assay accuracy.The spiked recovery experiment showed that the recoveries of colistin from feed,milk and meat samples were in the range of 77.83%to 113.38%with coefficient of variations less than 13%by ELISA analysis and less than 18%by LFIA analysis,respectively.Furthermore,actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental analysis.Conclusions:The developed assays can be used for rapid qualitative or quantitative detection of colistin in animal feed and food.

AB023.Ultrastable gold nanoparticles as a drug delivery system for ocular therapy

Background:Ocular therapy administrated by ophthalmic drops is advantageous thanks to its simplicity.However,efficiency of active molecules is limited when administered by this method.Indeed,more than 99.9%is discarded due to multiple factors including lacrimal drainage.Low retention time of drugs at the cornea leads to their poor penetration.Our hypothesis is that a drug delivery system based on gold nanoparticles should enhance the efficiency of the drugs.The main objective is to develop new methods to improve active molecules biodisponibility in ocular therapy thanks to a new drug delivery system implying gold nanoparticles.The specific objectives are:(I)to synthesize and characterize ultrastable gold nanoparticles,(II)to establish the drug encapsulation protocol,(III)to develop a separation method of free and encapsulated drugs to allow their quantification,(IV)to study the cytotoxicity of our gold nanoparticles.Methods:Ultrastable gold nanoparticles were synthesized by a new method and their ultrastability toward several harsh conditions was characterized.An encapsulation protocol was settled for several drugs.The separation of free and encapsulated drugs was performed with magnetic beads.The quantification of the encapsulated drugs was performed by HPLC.A MTS assay was performed on 3 corneal epithelial cell populations,exposed or unexposed to gold nanoparticles.Reconstructed corneas were prepared using the self-assembly method.A wound healing experience was performed on those corneas with or without nanoparticles.Results:Gold nanoparticles were synthesized and purified according to our new experimental conditions.They support harsh conditions as several cycles of freeze-drying,heating,salt exposition and ultracentrifugation.For the first time in literature,gold nanoparticle support autoclave sterilisation.The separation method involving magnetic beads was optimized to get rid of non-specific interactions.The encapsulation efficiency varies according to the active molecule.The MTS assay did not show diminution of the cellular viability when in presence of gold nanoparticles.Furthermore,gold nanoparticle exposition did not slow the wound healing of reconstructed corneas.Conclusions:Our new ultrastable gold nanoparticles can have a major impact in nanomedicine.They can support harsh conditions,as autoclave treatment,allowing their sterilisation for in vivo use.We showed that active molecules can be encapsulated in gold nanoparticles.In addition,they do not seem to cause any diminution of cellular viability.These data suggest the possible improvements in ocular therapy thanks to gold nanoparticles.

Gold Nanoparticles: Synthesis and Effect on Viability of Human Non-Small Lung Cancer Cells

Gold nanoparticles recently showed great interest for many uses including food, drug and medical applications. The algae Undaria sp. well known as wakame in South Asia are considered to be large edible brown algae. It provides nutritious source of dietary fiber, vitamin Bs and mineral. The present study aimed to investigate the use of Undaria sp. for green synthesis of metallic gold nanoparticles. The synthesized nanoparticles were characterized for physicochemical properties including size measurement and tested in vitro for their effect on viability of human non-small lung cancer H-460 cell line using the MTT assay. From the results, brown algae were able to chemically form nanoparticles with chloroauric acid solution possibly due to the sulphated polysaccharides found in algae. The particle sizes were found to be approximately 10 nm. The gold nanoparticles stabilized by the algae could decrease the cancer cell viability. However, the properties and biological activity of nanoparticles seemed to depend upon reaction time and temperature. Conclusively, gold nanoparticles synthesized and stabilized by the algae could decrease the cancer cell viability, thus indicating the potential of such nanoparticles for further study for anticancer activity.

A novel colloidal gold immunochromatography assay strip for the diagnosis of schistosomiasis japonica in domestic animals

Background:Schistosomiasis remains a major public health concern in China and an epidemiological survey has revealed that schistosome-infected bovines and goats are the main transmission sources for the disease.Therefore,development of a sensitive technique for the diagnosis of schistosomiasis in domestic animals is necessary.Method:A novel colloidal gold immunochromatography assay(GICA)strip was developed for detecting Schistosoma japonicum in domestic animals.The colloidal gold was conjugated with recombinant streptococcal protein G(rSPG).As the test and control lines,the schistosome soluble egg antigen and rSPG,respectively,were blotted on nitrocellulose membrane.Results:The lowest detectable serum dilution was 1∶640 for schistosome-infected buffaloes.The cross-reaction rate of GICA was 14.29%with Paramphistomum sp.in buffaloes,16.67%with Haemonchus sp.in goats,and 33.33%with Orientobilharzia sp.in goats.These results were slightly lower and similar to those obtained through ELISA.Moreover,the strips for detecting S.japonicum in mice,rabbits,buffaloes,and goats showed high sensitivity(100.00%,100.00%,100.00%,and 100.00%,respectively)and specificity(100.00%,100.00%,94.23%,and 88.64%,respectively).And the sensitivity or specificity of the GICA strips did not present any significant differences after storage for 12 months at room temperature.When compared with ELISA,the GICA strips exhibited similar sensitivity and specificity in the diagnosis of schistosomiasis in mice,rabbits,buffaloes,and goats.Besides,only 5μl of serum are required for the test and the detection can be completed within 5 min.Conclusion:This study is the first to develop a GICA strip using gold-rSPG conjugate for the diagnosing of schistosomiasis in domestic animals,and preliminary results showed that the developed strip may be suitable for large-scale screening of schistosomiasis in endemic areas.

Triple lines gold nanoparticle-based lateral flow assay for enhanced and simultaneous detection of Leishmania DNA and endogenous control

A novel triple lines lateral-flow assay （LFA） with enhanced sensitivity for the detection of Leishmania infantum DNA in dog blood samples was designed and successfully applied. The enhanced LFA methodology takes advantage of the gold nanoparticle tags （AuNPs） conjugated to polyclonal secondary antibodies, which recognize anti-FITC antibodies. The polyclonal nature of the secondary antibodies allows for multiple binding to primary antibodies, leading to enhanced AuNP plasmonics signal. Furthermore, endogenous control consisting of the amplified dog 18S rRNA gene was introduced to avoid false negatives. Using this strategy, 0.038 spiked Leishmania parasites per DNA amplification reaction （1 parasite/100 μL of DNA sample） were detected. Detection limit of LFA was found to be lower than that of the conventional techniques. In summary, our novel LFA design is a universal and simple sensing altemative that can be extended to several other biosensing scenarios.

Study on Rapid Detection of Tetracyclines,Fluoroquinolones and Sulfonamides in Milk

Aiming at the market demand for rapid detection of tetracyclines,fluoroquinolones and sulfonamides in milk,a golloidal gold immunochromatography test strip for simultaneous detection of tetracyclines,fluoroquinolones and sulfonamides in milk was prepared based on the principle of competitive inhibition immunochromatography. The performance indicators of the test strip were verified. The results showed that the test strip can simultaneously detect 4 tetracyclines,13 fluoroquinolones and 13 sulfonamides,and the detection limits all can meet the national residue limits; the tests strip exhibited false positive rate≤5% and false negative rate = 0; and no cross-reaction with other drugs was commonly found in milk,indicating good specificity. The method is simple,rapid,and has low cost and easy popularization. It provides a means for realizing on-site rapid detection and is of important practical significance to guarantee of safety of milk and dairy products in China.

黄金矿山企业化验室检测质量控制方法

黄金矿山企业化验室准确可靠的检测结果可为企业生产提供有效指导。简述了黄金矿山企业化验室在实施检测质量控制活动中,关于质量控制要求、质量控制方法、质量控制数据统计方法等3个方面的内容,以期对黄金矿山企业化验室做好质量控制工作提供借鉴。

Super-assembly of integrated gold magnetic assay with loopmediated isothermal amplification for point-of-care testing

With the increasing global threat of various diseases and infections,it is essential to develop a fast,low-cost,and easy-to-use point-of-care testing(POCT)system for inspections at all levels of medical institutions and self-examination at home.In this work,gold magnetic nanoparticles(GMNPs)are used as the key material,and a rapid visual detection method is designed through integrating loop-mediated isothermal amplification(LAMP)and lateral flow assay(LFA)biosensor for detecting a variety of analytes which includes whole blood,buccal swabs,and DNA.It is worth to note that the proposed method does not need DNA extraction.Furthermore,uracil DNA glycosylase(UDG)is employed to eliminate carrier contamination for preventing false positive results.The whole detection process can be finished within 25 min.The accuracy of detection is measured by assessing the polymorphisms of the methylenetetrahydrofolate reductase(MTHFR)C677T.The detection limit of the newly developed extraction-free detection system for MTHFR C677T is 0.16 ng/μL.A preliminary clinical study of the proposed method is carried out by analyzing 600 clinical samples(including 200 whole blood samples,100 buccal swabs,and 300 genomic DNA samples).The results indicate that the proposed method is 100%consistent with the sequencing results which provides a new choice for POCT and shows a broad application prospect in all levels of medical clinics and at home.

Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin A and zearalenone in corn, wheat, and feed samples

A one-step dual flow immunochromatographic assay （DICGA）, based on a competitive format, was de- veloped for simultaneous quantification of ochratoxin A （OTA） and zearalenone （ZEN） in corn, wheat, and feed sam- ples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53-12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06-39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry （LC-MS/MS） and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.

A homogeneous DNA assay by recovering inhibited emission of rare earth ions-doped upconversion nanoparticles

Robust and easy-to-use kits specific for a particular DNA sequence are desirable for early detection of diseases. However, the major challenge with these tests is often the background fluorescence artifacts arising from biological species due to employing UV and visible range of light. Here, we have reported a near-infrared (NIR) fluorescence "turn-on" kit based on rare earth ions doped nanoparticles, upconversion nanoparticles (UCNPs), and gold nanoparticles (AuNPs), which forms a fluorescence-quencher pair,brought together by a hairpin structure through the formation of double-stranded DNA (dsDNA), with quenched upconversion luminescence. In the presence of analytes, the molecular beacon opens to push AuNPs away from UCNPs, with a distance longer than the efficient quenching distance, so that the inhibited upconversion emission will be restored. We demonstrated that this assay provides a homogeneous, facile, simple and highly selective HIV-1 based DNA detection system with restore efficiency up to 85%, and the detection limit of 5 nm.

NanoHDA： A nanoparticle-assisted isothermal amplification technique for genotyping assays

Isothermal methods, such as helicase-dependent amplification （HDA）, have an advantage over polymerase chain reaction for DNA amplification owing to their ease of operation. Here, we developed a new HDA method that is nanoparticle-assisted, termed nanoHDA. This method uses gold nanoparticles （AuNPs） to improve the sensitivity and specificity of the isothermal method. In HDA, the denaturation of DNA templates is mediated by helicases, but this method is limited by the low denaturation efficiency of helicases. In this report, AuNPs with preferential affinity for single-stranded DNA （ssDNA） were utilized to improve the denaturation efficiency of helicases. The same affinity property of nanoparticles can also enhance specificity by suppressing primer-dimer formation. This nanoHDA method was employed to genotype the KRAS gene in genomic DNA samples from colorectal cancer patients, as achieved by the hybridization of nanoHDA amplicons using the NanoBioArray chip.

Visual detection of tropomyosin,a major shrimp allergenic protein using gold nanoparticles(AuNPs)-assisted colorimetric aptasensor

A gold nanoparticle-based label-free colorimetric assay was developed to detect the shrimp allergenic protein tropomyosin(TM),an important biomarker responsible for severe clinical reactivity to shellfish.In a gold nanoparticles(AuNPs)-tropomyosin-binding aptamer(TMBA)complex,the aptamer adsorbs onto the surface of AuNPs and dissociates in the presence of TM.In addition,AuNPs tend to aggregate in the presence of ionic salt,revealing a color change(i.e.,wine-red to purple/blue)with a shift in the maximum absorption peak from 520 nm.In the presence of specific binding TM,the aptamer folds into a tertiary structure where it more efficiently stabilizes AuNPs toward the salt-induced aggregation with a hypsochromic shift in the absorption spectra compared to the stabilized AuNPs by aptamer alone.Based on the aggregation and sensitive spectral transformation principle,the AuNPs-based colorimetric aptasensor was successfully applied to detect TM with a range of 10-200 nmol/L and a low detection limit of 40 nmol/L in water samples.The reliability,selectivity,and sensitivity of the aptasensor was then tested with food samples spiked with TM.The observed detection limit was as low as 70 nmol/L in shrimp,90 nmol/L in tofu,and 80 nmol/L in eggs,respectively.We anticipate the proposed AuNPs-based colorimetric aptasensor assay possesses a high potential for the easy and efficient visual colorimetric detection of TM.

An immunochromatographic assay for rapid and simultaneous detection of levonorgestrel and methylprednisolone in water samples

Synthetic contraceptive levonorgestrel （LNG） and glucocorticoid methylprednisolone （MP） residues are eventually discarded to environmental water system and function as environmental hormones, displaying potential risk to humans and ecosystems, thus there is an urgent need for fast, sensitive and simultaneous detection of these compounds in water samples. In this study, a competitive immunochromatographic assay （ICA） using colloidal gold-labeled polyclonal antibodies as probes for rapid and simultaneous detection of LNG and MP in water samples was developed. The visual detection limits of LNG and MP in water samples were 10 ng/mL. The detection process could be completed within 10 min. There was no cross-reactivity of the ICA with other seven compounds. The strips could be stored at 4 ℃ for 10 weeks without significant loss of activity. The assay is a suitable tool for rapid and semi- quantitative detection of LNG and MP in water samples on site.

Colorimetric detection of glucose using a boronic acid derivative receptor attached to unmodified AuNPs

A simple, cheap and non-enzymatic colorimetric strategy for glucose detection has been designed based on the interactions between a phenylboronic acid （PBA） derivative, which is coupled with gold nanoparticles （AuNPs） as the colorimetric reporters, and glucose. The PBA-AuNPs hybrid system proposed here exhibits ordered photochemistry behaviors upon the addition of glucose at different pH values. There are two linear regions of glucose concentration for the glucose sensor at different pH values, i.e., between 0.1 mmol/L and 9.8 mmol/L at pH 6 with the detection limit of 64μmol/L and between 0 and 6.5 mmol/L with the detection limit of 48 μmol/L at pH 9, respectively. To test the practicality of the sensor system, we also applied this assay to detect a glucose sample in the artificial saliva.

Development of a Handheld Nano-centrifugal Device for Visual Virus Detection

Gold nanoparticles(AuNPs)colorimetric assays based on distance-dependent optical characteristics have been widely employed for bioanalysis.However,this assay is not effective for visually detecting low-concentration targets due to the faint color change.Here,we developed a handheld nano-centrifugal device which could separate the crosslinked and non-crosslinked AuNPs.Results showed that the handheld nano-centrifugal device could easily reach more than 6000 r/min within 10 s simply by stretching and tightening the coiled rope in an appropriate rhythm.Further,combined with the CRISPR/Cas12a nucleic acids recognition system,a field-deployable colorimetric platform termed handheld nano-centrifugal device assisted CRISPR/Cas12a(Hand-CRISPR)has been validated.Moreover,clinical diagnostics applications for Epstein-Barr virus(EBV)and severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)detection with high sensitivity and accuracy(100%consistency with reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR)test results)have been demonstrated.Overall,the Hand-CRISPR platform showed great promise in point-of-care-test(POCT)application,expected to become a powerful supplement to the standard nucleic acid testing method in remote or poverty-stricken areas.