Development of a Dual Fluorescence Signal-Enhancement Immunosensor Based on Substrate Modification for Simultaneous Detection of Interleukin-6 and Procalcitonin.

High-sensitivity detection of biomarkers is of great significance to improve the accuracy of disease diagnosis and the rate of occult disease diagnosis. Using a substrate modification and two-color quantum dot (QD) nanobeads (QBs), we have developed a dual fluorescence signal-enhancement immunosensor for sensitive, simultaneous detection of interleukin 6 (IL-6) and procalcitonin (PCT) at low volumes (∼20 µL). First, the QBs compatible with QDs with different surface ligands were prepared by optimizing surfactants based on the microemulsion method. Through the use of a fluorescence-linked immunosorbent assay (FLISA), the feasibility of a dual signal-enhancement immunosensor was verified, and a 5-fold enhancement of fluorescence intensity was achieved after the directional coating of the antibodies on sulfhydryl functionalization (-SH) substrates and the preparation of QBs by using a polymer and silica double-protection method. Next, a simple polydimethylsiloxane (HS-PDMS) immunosensor with a low volume consumption was prepared. Under optimal conditions, we achieved the simultaneous detection of IL-6 and PCT with a linear range of 0.05-50 ng/mL, and the limit of detection (LOD) was 24 and 32 pg/mL, respectively. The result is comparable to two-color QBs-FLISA with a sulfhydryl microplate, even though only 20% of its volume was used. Thus, the dual fluorescence signal-enhancement HS-PDMS immunosensor offers the capability of early microvolume diagnosis of diseases, while the detection of inflammatory factors is clinically important for assisting disease diagnosis and determining disease progression.

Tailored three-color quantum dots nanobeads for multiplexed detection with tunable detection range and multilevel sensitivity of signal-amplified immunosensor.

The excellent optical properties of quantum dots (QDs) make them as an ideal fluorescent probe for multiplexed detection, however, the interference between different emission spectra, the dependence of excitation wavelengths, and the sharp decrease of quantum yield (QY) during surface modification are issues that cannot be ignored. Herein, a dual protection scheme of polymer and silica was proposed to prepare high-quality three-color QDs nanobeads using QDs with different ligands. In comparison with single-core QDs, the fluorescence signal of the prepared QD nanobeads (QBs) is increased by more than 1,000 times and has better stability. Considering the excitation efficiency of QDs, we tailor three-color QBs as fluorescent probes based on fluorescence-linked immunosorbent assays (tQBs-FLISA) to detect multiple inflammatory biomarkers simultaneously with tunable detection ranges. This resulted in highly sensitive detection of three inflammatory biomarkers in comparison to the single-core QD-FLISA, the sensitivities of C-reactive protein (CRP), serum amyloid A (SAA), and procalcitonin (PCT) were increased by 16-fold, 19-fold, and 5-fold, respectively, to 0.48 ng/mL, 0.42 ng/mL, and 10 pg/mL. Furthermore, the tQBs-FLISA showed good accuracy without interference from common serum factors. In this strategy, a three-color QBs suitable for multilevel sensitivity and tunable detection range was tailored using the versatile polymer and silica dual protection method, building high-performance immunosensor for in vitro diagnostics (IVD).

Tailored different sizes of quantum dot nanobeads for sensitive and quantitative detection based on the competition fluorescence-linked immunosorbent assay platform.

Highly stable and multicolor photoluminescent (PL) quantum dots (QDs) have attracted widespread attention as ideal probe materials in the field of in vitro diagnostics (IVD), especially the fluorescence-linked immunosorbent assay (FLISA), due to their advantages of high-throughput, high stability, and high sensitivity. However, the size of QDs as fluorescent probes have significant effects on antigen-antibody performance. Therefore, it is critical to design suitable QDs for obtain excellent quantitative detection-based biosensors. In this paper, we prepared different sizes of aqueous QDs (30 nm, 116 nm, 219 nm, and 320 nm) as fluorescent probes to optimize the competitive FLISA platform. The SARS-CoV-2 neutralizing antibody (NTAB) assay was used as an example, and it was found that the size of the QDs has a significant impact on the antigen-antibody binding efficiency and detection sensitivity in competitive FLISA platform. The results showed that these QD nanobeads (QBs, ∼219 nm) could be used as a labeled probe for competitive FLISA, with half-maximal inhibitory concentration (IC50) of 1.34 ng/mL and limit of detection (LOD) of 0.21 pg/mL for NTAB detection. More importantly, the results showed good specificity and accuracy, and the QB219 probe was able to efficiently bind NTAB without interference from other substances in the serum. Given the above advantages, the nanoprobe material (∼200 nm) offers considerable potential as a competitive FLISA platform in the field of IVD.