Highly Efficient Purification Of Quantum Dot-Conjugates And Quantum Dots/Quantum Dot Beads Based Immunochromatographic Strip For Folic Acid Detection

Lateral flow immuno-chromatographic assay (LFIA) is a popular screening tool for onsite testing because of its rapidity, acceptable anti-interference ability, and user-friendliness. Compared with traditional colloidal gold nanoparticles (AuNPs), fluorescent nanoparticles (FNs) possess a relatively high sensitivity and have been widely used to the quantitative detection. FNs-based ICTS assay is represenitive fluorescence immuno sensor of increasing research focus and have attracted intensive attention in recent years. Organic dyes, a type of fluorescent-based sensor, are useful tools for ICTS assay. In comparison to traditional organic dye, quantum dots (QDs) are regarded as promising fluorescent probes due to their unique properties (e.g. size-tunable emission, broad adsorption, narrow and symmetric photoluminescence spectra, strong luminescence, and robust photostability). Conjugation of QDs to biomacromolecules and purification of QD-based conjugates are key element to immunological diagnosis. At present, the controllable bioconjugation procedures has been reported many times in the scientific literature but the purification of QDs-based conjugates was still a difficulity situation because of operating complex, low-recovery rate, and non-scale operation etc. Thus, the key point to realize industrialization application of QDs is development of the simple and efficient purification method for QDs-based conjugates.A new surface modification concept using D-(+)-glucosamine covalently attracted on carboxylated QDs (QD620) is developed to decrease the surface potential of the originally QDs, which makes QDs’fast precipitation and high efficient purified recovery. Taken anti-AFB1 monoclonal antibody (anti AFB1 mAb) and streptavidin (SA) as model proteins, we prepared QD-mAb and QD-SA conjugates with different molar ratio then covered the QDs with D-(+)-glucosamine. Adjusted the solution pH nearing the conjugates’isolectric point (around pH4.5) and centrifuged it at 20000 rpm (29000 g) for 30 min for purification. The characterization of resulting QD based conjugates were evaluated by enzyme linked immunosorbent assay (ELISA), argarose gel electrophoresis, dynamic light scattering (DLS), fluoro-photometric analysis and immuno-chromatographic assay (ICA), the results showed that D-(+)-glucosamine blocked QD-mAb and QD-SA conjugates purified through centrifuge method kept good QDs’ fluorescence, biological activity, particles’ mono-dispersity and colloidal stability.The other part of work was focused on establishing quantum dots or quantum dot beads (QDB) based immuno-chromatographic test strip for quantitative detection of folic acid (FA). Four types of strip with different labeling pattern including QD-anti FA mAb, QD-SA-biotin-mAb, QD-BSA-FA and QDB-BSA-FA, respectively, were prepared and the strip labeled with QDB-BSA-FA posesses the best detection limit. An orthogonal L9(3)3 test design was used to optimize the best parameters, which were as follows:QDB-FA conjugate ratio was 1:100, QDB concentration on conjugate pad was 7 nmol/L and anti-FA mAb concentration on test line was 0.5 mg/mL. In PBS matrix, the quantitative detecting standard curve of the QDB-FA based strip was y=-0.1160Ln(x)+0.8924 with a reliable coefficient of correlation (R2=0.9590). The linear range was between 1 ng/mL to 50 ng/mL and the half maximal inhibitory concentration (IC50) was 29.35 ng/mL. In simulated negative human serum samples, the quantitative detecting standard curve was y=-0.1744Ln(x)+1.1044 with a reliable coefficient of correlation (R2=0.9683). The linear range was 3-50 ng/mL, and IC50 was 32.00 ng/mL. The detection limit for simulated human serum was 3.54 ng/mL and the recovery of spiked FA testing was between 95.86% to 102.93%. The coefficient variation of the same batch of strips showed 5.51% to 11.55% in intra-assays in while it reached 6.66% to 10.58% in inter-assays, indicated the strip had good accuracy and precision. This strip had severe cross activity with folic acid analogue including pteroic acid, dihydrofolic acid and tetrahydrofolic acid, which was much suitable for broad spectrum detection.