Jinchuan Liu

Improving sensitivity of gold nanoparticle based fluorescence quenching and colorimetric aptasensor by using water resuspended gold nanoparticle

Gold nanoparticles (AuNPs) based fluorescence quenching or colorimetric aptasensor have been developed for many analytes recently largely because of the ease of detection, high sensitivity, and potential for high-throughput analysis. However, the effects of remnant non-AuNPs components in the colloid gold solution on these assays performance remain unclear. For the first time, we demonstrated that the remnant sodium citrate and the reaction products of three acids play counteractive roles in AuNPs based fluorescence quenching and colorimetric aptasensor in three ways in this study. First, the remnant sodium citrate in the colloid gold solution could increase the fluorescence intensity of FAM labeled on the aptamer that reduce the efficiency of AuNPs fluorescent quenching. Second, the reaction products of citric acid, HCl and ketoglutaric acid reduce the fluorescence recovery by quenching the fluorescence of FAM labeled on the aptamer dissociated from the surface of AuNPs upon addition of target. Lastly, the reaction products of three acids reduce the pH value of the colloid gold solution that reduce the sensitivity of AuNPs based colorimetric aptasensor by increasing the adsorption of aptamer to surface of AuNPs. With sulfadimethoxine and thrombin as model analytes, we found that water resuspended AuNPs can significantly increase the sensitivity by more than 10-fold for AuNPs based fluorescence quenching aptasensor. In the AuNPs based colorimetric aptasensor for sulfadimethoxine using the water resuspended AuNPs, the sensitivity also was increased by 10-fold compared with that of original AuNPs. The findings in this study provide theoretical guidance for further improving AuNPs based fluorescent quenching and colorimetric aptasensor by adjusting the composition of AuNPs solution.

Highly sensitive colorimetric detection of 17β-estradiol using split DNA aptamers immobilized on unmodified gold nanoparticles

Gold nanoparticle (AuNP) based colorimetric aptasensor have been developed for many analytes recently largely because of the ease of detection, high sensitivity, and potential for high-throughput analysis. Most of the target aptamers for detection have short sequences. However, the approach shows poor performance in terms of detection sensitivity for most of the long-sequence aptamers. To address this problem, for the first time, we split the 76 mer aptamer of 17β-estradiol into two short pieces to improve the AuNP based colorimetric sensitivity. Our results showed that the split P1 + P2 still retained the original 76 mer aptamers affinity and specificity but increased the detection limit by 10-fold, demonstrating that as low as 0.1 ng/mL 17β-estradiol could be detected. The increased sensitivity may be caused by lower aptamer adsorption concentration and a lower affinity to the AuNPs of a short single-strand DNA (ssDNA) sequence. Our study provided a new way to use long-sequence aptamers to develop a highly sensitive AuNP-based colorimetric aptasensor.

A simple and sensitive approach for ochratoxin A detection using a label-free fluorescent aptasensor.

Ochratoxin A(OTA) is found to be one of the predominant contaminating mycotoxins in a wide variety of food commodities. To avoid the risk of OTA consumption, the detection and quantitation of OTA level are of great significance. Based on the fact that ssDNA aptamer has the ability to form a double-strand structure with its complementary sequence, a simple and rapid aptamer-based label-free approach for highly sensitive and selective fluorescence detection of OTA was developed by using ultra-sensitive double-strand DNA specific dyes PicoGreen. The results showed that as low as 1 ng/mL of OTA could be detected with a dynamic range of more than 5 orders of magnitude which satisfies the requirements for OTA maximum residue limit in various food regulated by European Commission. With the specificity of aptamer, the assay exhibited high selectivity for OTA against two other analogues (N-acetyl-l-phenylalanine and zearalenone). We also tested the aptasensor practicability using real sample of 1% beer spiked with a series of concentration of OTA and the results show good tolerance to matrix effect. All detections could be achieved in less than 30 min, which provides a simple, quick and sensitive detection method for OTA screening in food safety and could be easily extend to other small molecular chemical compounds detection which aptamer has been selected.

High sensitive rapid visual detection of sulfadimethoxine by label-freeaptasensor

Quickly and sensitively detection of antibiotic residues in animal products often requires time-consuming techniques and expensive instrumentation. To address these limitations, a high sensitive aptasensor for sulfadimethoxine (SDM) using unmodified gold nanoparticles (AuNPs) was developed in the present study. The assay sensitivity was improved by optimizing several key parameters such as the amount of aptamer adsorbed to AuNPs, the concentration of salt and the AuNPs pH value. To our knowledge, this is the first time to investigate the effect of AuNPs pH value on the sensitivity of aptamer-AuNPs based label-free assay. The best procedure for SDM analysis in our system was: the pH value at 8.0, and the concentration of aptamer at 2 μM, and the concentration of salt at 2 M. The linear range for the current analytical system was from 50 ng/mL to 1 μg/mL, and the detection limit was 50 ng/mL, which is lower than the Maximum Residue Limit (MRL) (100 ng/mL) in edible animal tissues regulated by China and European commission. Our study provides a simple, fast, and easy to read method for SDM analysis with high sensitivity, which can be applied in future on-site detection in animal products.

Highly sensitive fluorescent detection of small molecules, ions, and proteins using a universal label-free aptasensor.

A facile and universal aptamer-based label-free approach for highly selective and sensitive fluorescence detection of a broad range of targets including small molecules, inorganic ions and proteins was developed by using PicoGreen to transduce the fluorescent signal of the double stranded DNA duplex formed between a free aptamer and its complementary strand.

Gold nanoparticle–based colorimetric aptasensor for rapid detection of six organophosphorous pesticides

Fast immunoassay-based screening methods are unavailable for most small-molecule pesticides because of a lack of immunogenicity and the difficulty in obtaining antibodies by animal immunization. Aptamers are single-stranded DNA molecules selected through an in vitro process, which can bind to any target including nonimmunogenic small molecules with high affinity and specificity. Although various aptamer-based sensing methods have been developed for antibiotics, microorganisms, heavy metal ions, and biotoxins, there are few reports on aptamer-based methods for quick detection of organophosphorous pesticides. The gold (Au) nanoparticle (AuNP) colorimetric assay is a widely utilized rapid detection method because of properties such as easy operation and visualized results. In the present study, organophosphorous pesticide aptamers were adsorbed on the surface of AuNPs to stabilize the AuNP solution against high concentrations of salt to prevent AuNP aggregation. After the addition of targets, the aptamers binding to the targets are detached from the AuNPs, resulting in aggregation of AuNPs and a color change from red to purple-blue. The proposed method can detect 6 organophosphorous pesticides with good recoveries from 72% to 135% in environmental river water samples. The present study provides a new way for simple, rapid, and multiplex detection of organophosphorous pesticides.

Colorimetric aptasensor using unmodified gold nanoparticles for homogeneous multiplex detection.

Colorimetric aptasensors using unmodified gold nanoparticles (AuNPs) have attracted much attention because of their low cost, simplicity, and practicality, and they have been developed for various targets in the past several years. However, previous research has focused on developing single-target assays. Here, we report the development of a homogeneous multiplex aptasensor by using more than one class of aptamers to stabilize AuNPs. Using sulfadimethoxine (SDM), kanamycin (KAN) and adenosine (ADE) as example targets, a KAN aptamer (750 nM), an SDM aptamer (250 nM) and an ADE aptamer (500 nM) were mixed at a 1∶1∶1 volume ratio and adsorbed directly onto the surface of unmodified AuNPs by electrostatic interaction. Upon the addition of any of the three targets, the conformation of the corresponding aptamer changed from a random coil structure to a rigid folded structure, which could not adsorb and stabilize AuNPs. The AuNPs aggregated in a specific reaction buffer (20 mM Tris-HCl containing 20 mM NaCl and 5 mM KCl), which led to a color change from red to purple/blue. These results demonstrate that the multiplex colorimetric aptasensor detected three targets simultaneously while maintaining the same sensitivity as a single-target aptasensor for each individual target. The multiplex aptasensor could be extended to other aptamers for various molecular detection events. Due to its simple design, easy operation, fast response, cost effectiveness and lack of need for sophisticated instrumentation, the proposed strategy provides a powerful tool to examine large numbers of samples to screen for a small number of potentially positive samples containing more than one analyte, which can be further validated using sophisticated instruments.

AuNP‐based colorimetric aptasensor for rapid detection of six organophosphorus pesticides

Fast immunoassay-based screening methods are unavailable for most small-molecule pesticides because of a lack of immunogenicity and the difficulty in obtaining antibodies by animal immunization. Aptamers are single-stranded DNA molecules selected through an in vitro process, which can bind to any target including nonimmunogenic small molecules with high affinity and specificity. Although various aptamer-based sensing methods have been developed for antibiotics, microorganisms, heavy metal ions, and biotoxins, there are few reports on aptamer-based methods for quick detection of organophosphorous pesticides. The gold (Au) nanoparticle (AuNP) colorimetric assay is a widely utilized rapid detection method because of properties such as easy operation and visualized results. In the present study, organophosphorous pesticide aptamers were adsorbed on the surface of AuNPs to stabilize the AuNP solution against high concentrations of salt to prevent AuNP aggregation. After the addition of targets, the aptamers binding to the targets are detached from the AuNPs, resulting in aggregation of AuNPs and a color change from red to purple-blue. The proposed method can detect 6 organophosphorous pesticides with good recoveries from 72% to 135% in environmental river water samples. The present study provides a new way for simple, rapid, and multiplex detection of organophosphorous pesticides.

A simple and sensitive label-free fluorescent approach for protein detection based on a Perylene probe and aptamer

Highly sensitive detection of proteins is of great importance for effective clinical diagnosis and biomedical research. However, so far most detection methods rely on antibody-based immunoassays and are usually laborious and time-consuming with poor sensitivity. Here, we developed a simple and ultra-sensitive method to detect a biomarker protein-thrombin by taking advantage of the fluorescent probe Perylene tetracarboxylic acid diimide (PTCDI) derivatives and thrombin aptamer. The water-soluble dye PTCDI shows strong fluorescence in buffer solution for the existence of free dye monomer, but becomes weak after aggregation through self-assembly on nucleic acid aptamer. In the presence of thrombin, it specifically binds to thrombin aptamer which causes the conformational transition between aptamer and PTCDI and results in a significant fluorescence recovery. The results showed that as low as 40 pM of thrombin could be detected by this method. The high sensitivity of the developed sensing system mainly attributes to the ultra-sensitivity of the fluorescence intensity changes of PTCDI. With the specificity of aptamer, the assay exhibited high selectivity for thrombin against three other proteins (bovine serum albumin, lysozyme, mouse IgG) and 1% diluted fetal bovine serum. The detection method might be extended to sensitive detection of a variety of proteins for its advantages of isothermal conditions required, simple and rapid without multiple separation and washing steps.

Identification and quantitation of phenolic compounds from the seed and pomace of Perilla frutescens using HPLC/PDA and HPLC-ESI/QTOF/MS/MS.

INTRODUCTION Perilla frutescens (L.) Britt., an essential traditional Asian crop and Chinese medicine, potentially exerts anti-oxidation effects through its phenolic compounds. These compounds have already been reported in perilla seed, however, little is reported in Perilla pomace, the primary waste during oil production of Perilla seed. OBJECTIVE To investigate major phenolic compounds in perilla seeds and pomaces in order to check whether the pomace could be an alternative resource to the seed for nutritional and medical purposes. METHODS Compounds in extracts of perilla seeds and pomaces were separated by high-performance liquid chromatography and detected by photodiode array, and by electrospray ionisation with quadrupole time-of-flight tandem mass spectrometry. Herb-markers selected by principal components analysis were then quantified in both seeds and pomaces. Moreover, a fingerprinting approach and multiple discriminant analysis were applied to screen the phenolic markers in 22 samples. RESULTS Ten phenols were tentatively identified, among which four (rosmarinic acid, luteolin, apigenin and rosmarinic acid-3-O-glucoside) were selected as herb-markers. Perilla seeds and pomaces showed similar phenol profiles, however, the pomaces contained almost two times the amount of the four herb-markers than the seeds. CONCLUSION The results indicated perilla pomace is a promising alternative source of phenolic compounds that could be recovered and potentially used as natural anti-oxidants.