Salbutamol (SAL) is known as quick-acting β-2 receptor agonist and its use in humans for pulmonary diseases and/or in animal feed is limited because of associated potential hazardous effects on health. Several techniques are available for the detection of SAL, but are expensive and time consuming. Here for the first time, a novel pre-assembled DNA immunoprobe-based immuno-PCR assay was developed to investigate the levels of SAL in human urine samples and compared the proposed immuno-PCR assay for the detection of SAL with that of direct competitive ELISA. Under the optimized conditions, the assay showed a linear range over seven orders of magnitude, whereas the limit of detection of SAL in buffer was found to be 21 fg/mL. Current immuno-PCR assay exhibited a 300-fold better detection limit for SAL as compared to one achieved with direct competitive ELISA using the same antibody. In conclusion, it has been found that the developed method is highly sensitive and simple method that can significantly enhance the detection sensitivity for small molecules. Moreover it can be modified and used for detecting other small molecules and/or chemicals that exist in the environment and are responsible for the environmental pollution.
Zhi Li,Chuqin Yang,Wenying Lu,Zhaohui Chu,Jingwen Zhang,Ming Li,Qingjiang Wang
It was desired to develop the ultrasensitive and efficient strategy for detecting ultratrace aflatoxin B 1 (AFB 1 ) and protecting consumers' health. In this study, a magnetic immuno-PCR method (MIPCR) based on the barcode DNA was described and evaluated for detecting AFB 1 in agricultural by-product samples. Under the optimal MIPCR , the limit of detection (LOD) was 0.01 pg mL −1 , the half inhibitive concentration (IC 50 ) was 1.72 pg mL −1 , and the detection range was between 0.01 and 100 pg mL −1 . The LOD and IC 50 showed 480-fold and 21-fold lower than that of ELISA , respectively. The sensitivity of MIPCR had reached an ultratrace level and considerably improved. The reasonable specificity, precision and accuracy had been demonstrated. Furthermore, the high reliability and applicability of the MIPCR had been further confirmed by the good correlation of LC-MS/MS for the authentic samples. The proposed MIPCR had significantly shortened the analytical procedure, testing time and workload through the powerful probes and magnetic separation, which indicated the high-efficiency performance. This study could provide an alternative approach to detect the AFB 1 contamination, and a critical reference for the ultrasensitive and efficient detection of other hazardous chemicals.
Chuan Zhang,Kaixin Zheng,Chi Li,Ranran Zhang,Yicheng Zhu,Linxiao Xia,Yicheng Ma,Hans M. Wyss,Xiaoyu Cheng,Sailing He
Detecting proteins in ultralow concentrations in complex media is important for many applications but often relies on complicated techniques. Herein, a single-molecule protein analyzer with the potential for high-throughput applications is reported. Gold-coated magnetic nanoparticles with DNA-labeled antibodies were used for target recognition and separation. The immunocomplex was loaded into microdroplets generated with centrifugation. Immuno-PCR amplification of the DNA enabled the quantification of proteins at the level of single molecules. As an example, ultrasensitive detection of α-synuclein, a biomarker for neurodegenerative diseases, is achieved. The limit of detection was determined to be ∼50 aM in buffer and ∼170 aM in serum. The method exhibited high specificity and could be used to analyze post-translational modifications such as protein phosphorylation. This study will inspire wider studies on single-molecule protein detection, especially in disease diagnostics, biomarker discovery, and drug development.
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