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.

Highly Sensitive, Stable InP Quantum Dot Fluorescent Probes for Quantitative Immunoassay Through Nanostructure Tailoring and Biotin-Streptavidin Coupling.

Nontoxic, highly sensitive InP quantum dot (QD) fluorescent immunoassay probes are promising biomedical detection modalities due to their unique properties. However, InP-based QDs are prone to surface oxidation, and the stability of InP QD-based probes in biocompatible environments remains a crucial challenge. Although the thick shell can provide some protection during the phase transfer process of hydrophobic QDs, the photoluminescence quantum yield (PLQY) is generally decreased because of the contradiction between lattice stress relaxation and thick shell growth. Herein, we developed thick-shell InP-based core/shell QDs by inserting a ZnSeS alloy layer. The ternary ZnSeS intermediate shell could effectively facilitate lattice stress relaxation and passivate the defect states. The synthesized InP/ZnSe/ZnSeS/ZnS core/alloy shell/shell QDs (CAS-InP QDs) with nanostructure tailoring revealed a larger size, high PLQY (90%), and high optical stability. After amphiphilic polymer encapsulation, the aqueous CAS-InP QDs presented almost constant fluorescence attenuation and stable PL intensity under different temperatures, UV radiation, and pH solutions. The CAS-InP QDs were excellent labels of the fluorescence-linked immunosorbent assay (FLISA) for detecting C-reactive protein (CRP). The biotin-streptavidin (Bio-SA) system was first introduced in the FLISA to further improve the sensitivity, and the CAS-InP QDs-based SA-Bio sandwich FLISA realized the detection of CRP with an impressive limit of detection (LOD) of 0.83 ng/mL. It is believed that the stable and sensitive InP QD fluorescent probes will drive the rapid development of future eco-friendly, cost-effective, and sensitive in vitro diagnostic kits.

Fabrication of CuInZnS/ZnS Quantum Dot Microbeads by a Two-Step Approach of Emulsification-Solvent Evaporation and Surfactant Substitution and Its Application for Quantitative Detection.

CuInS2 quantum dots (CIS QDs) are considered to be promising alternatives for Cd-based QDs in the fields of biology and medicine. However, high-quality hydrophobic CIS QDs are difficult to be transferred to water due to their 1-dodecylmercaptan (DDT) ligands. Therefore, the fluorescence and stability of the prepared aqueous CIS QDs is not enough to meet the requirement for sensitive detection. Here, as large as 13 nm CuInZnS/ZnS QDs with DDT ligands were first synthesized, and then, CuInZnS/ZnS microbeads (QBs) containing thousands of QDs were successfully fabricated by a two-step approach of emulsion-solvent evaporation and surfactant substitution. Through emulsion-solvent evaporation, the CuInZnS/ZnS QDs formed microbeads in the microemulsion with dodecyl trimethylammonium bromide (DTAB), and the Förster resonance energy transfer (FRET) has been effectively overcome. Then, CO-520 was introduced to substitute DTAB to improve the stability and water solubility. Lastly, the microbeads were coated with a SiO2 shell and carboxylated. Subsequently, the constructed QBs (∼210 nm) were used as labels in a fluorescence immunosorbent assay (FLISA) for quantitative detection of heart type fatty acid binding protein (H-FABP), and the limit of detection was 0.48 ng mL-1, which indicated a greatly improved detection sensitivity compared to that of the Cd-free QDs. The highly fluorescent and stable CuInZnS/ZnS QBs will have great application prospects in many biological fields.

Biomolecular Surface Functionalization and Stabilization Method to Fabricate Quantum Dots Nanobeads for Accurate Biosensing Detection.

The surface functionalization of quantum dots (QDs) is essential for their application as a label material in a biological field. Here, a protein surface functionalization approach was introduced to combine with silica encapsulation for the sustainable and stable synthesis of QDs nanobeads for biomarker detection. The formation of QDs nanobeads was achieved by multiple mercapto groups in bovine serum albumin (BSA) macromolecules as multidentate ligands to replace hydrophobic ligands on the surface of QDs and decompression. The resulting QDs nanobeads exhibited 20 times more photoluminescence than the corresponding hydrophobic QDs and presented excellent stability under physiological conditions due to the protection of BSA and silica. The nanobeads served as a robust signal-generating reagent to construct the lateral flow immunoassay (LFIA) biosensor for the detection of glycosylated hemoglobin (HbA1c). The concentration of HbA1c was determined within 10 min with high specificity using only 60 µL of whole blood samples collected clinically. The nanobeads-based LFIA biosensor exhibited linear detection of HbA1c from 4.2% to 13.6%. The accuracy and stability of this approach in clinical utility was demonstrated by the detection of HbA1c after a long-term storage of test strips. This protein surface modification technology provides a new way for improving the biological properties of QDs in clinical diagnosis.

Sensitive Immunoassay Based on Biocompatible and Robust Silica-Coated Cd-Free InP-Based Quantum Dots.

Low-toxic InP quantum dots (QDs) as an ideal candidate for Cd-based QDs have tremendous potential for next-generation commercial display and biological detection applications. However, the progress in biological detection is still far behind that of the Cd-based QDs. This is mainly because the InP-based QDs are of inferior stability and photoluminescence quantum yield (PL QY) in aqueous solution. Here, PL QY of 65% and excellent stability of InP/GaP/ZnS QD@SiO2 nanoparticles have been successfully synthesized via a silica coating method. The containing thiol-capped hydrophobic InP/GaP/ZnS QDs were pre-silanized with waterless, ammonia-free hydrolysis tetraethyl orthosilicate, and subsequently, an outer silica shell was generated in the reverse microemulsion. The corresponding QD-based fluorescence-linked immunosorbent assay exhibits a high sensitivity of 0.9 ng mL-1 for C-reactive protein and the broad detection range of 1-1000 ng mL-1, which was close to that of the state-of-the-art Cd-based QD@SiO2 nanoparticles and had the highest sensitivity of Cd-free QDs so far. This work provides a very successful silica coating method for the containing thiol-capped hydrophobic QDs and the QDs highly sensitive to water and oxygen, and the obtained InP/GaP/ZnS QD@SiO2 nanoparticles were considered as the robust, biocompatible, and promising Cd-free fluorescent labels for the further ultra-sensitive detection.

A quantum dot microspheres-based highly specific and sensitive three-dimensional microarray for multiplexed detection of inflammatory factors.

The development trend ofin vitrodiagnostics is to obtain various biological information from a sample at extremely low concentration and volume, which has promoted its progress in accurate and sensitive multiplexed detection. Here, we developed a single color quantum dot (QD) based three-dimensional (3D) structure matrix microarray and conducted the detection of two inflammatory factors (C-reactive protein (CRP) and serum amyloid A (SAA)) by a self-built fluorescence detection system. This strategy increased detection sensitivity by immobilizing the antibody specifically on the 3D substrate because it captured more than about 7 times of 'effective' antibodies compared to the two-dimensional (2D) plane. Compared to the dual QDs-2D fluorescence-linked immunosorbent assay, the limit of detection (LOD) of 3D microarray based on QDs modified with amphiphilic polymers has been further improved to 0.11 ng ml-1for SAA assay and to 0.16 ng ml-1for CRP assay, respectively. By using QD microspheres (SiO2@QDs@SiO2-COOH, containing approximately 200-300 hydrophobic QDs on per SiO2sphere) as fluorescent labels, the LOD for CRP and SAA of 3D microarray reached as high as 15 pg ml-1and 86 pg ml-1, and the sensitivity was further improved by 28-fold and 425-fold, respectively. Because of its excellent performance, this QD microspheres-based 3D microarray has great application potential for highly sensitive and multiplexed quantitative detection of other biomarkers, small molecules, and antibiotic residues in biomedicine and food safety.

Highly sensitive and accurate detection of C-reactive protein by CdSe/ZnS quantum dot-based fluorescence-linked immunosorbent assay.

BACKGROUND: The conventional and widely used enzyme-linked immunosorbent assays (ELISA), due to specificity and high-sensitivity, were suitable in vitro diagnosis. But enzymes are vulnerable to the external conditions, and the complex operation steps limit its application. Semiconductor quantum dots have been successfully used in biological and medical research due to the high photoluminescence and high resistance to photobleaching. In this study, we have developed a novel quantum dot-labeled immunosorbent assay for rapid disease detection of C-reactive protein (CRP). RESULTS: The assay for the detection of CRP can provide a wide analytical range of 1.56-400 ng/mL with the limit of detection (LOD) = 0.46 ng/mL and the limit of quantification = 1.53 ng/mL. The precision of the assay has been confirmed for low coefficient of variation, less than 10% (intra-assay) and less than 15% (inter-assay). The accuracy of assay meets the requirements with the recoveries of 95.4-105.7%. Furthermore, clinical samples have been collected and used for correlation analysis between this FLISA and gold standard Roche immunoturbidimetry. It shows excellent accurate concordance and the correlation coefficient value (R) is as high as 0.989 (n = 34). CONCLUSIONS: This in vitro quantum dot-based detection method offers a lower LOD and a wide liner detection range than ELISA. The total reaction time is only 50 min, which is much shorter than the commercialization ELISA (about 120 min). All of the results show that a convenient, sensitive, and accurate fluorescence-linked immunosorbent assay method has been well established for the detection of CRP samples. Therefore, this method has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.

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.

Recent advances in quantum dot-based fluorescence-linked immunosorbent assays.

Fluorescence immunoassays have been given considerable attention among the quantitative detection methods in the clinical medicine and food safety testing fields. In particular, semiconductor quantum dots (QDs) have become ideal fluorescent probes for highly sensitive and multiplexed detection due to their unique photophysical properties, and the QD fluorescence-linked immunosorbent assay (FLISA) with high sensitivity, high accuracy, and high throughput has been greatly developed recently. In this manuscript, the advantages of applying QDs to FLISA platforms and some strategies for their application to in vitro diagnostics and food safety are discussed. Given the rapid development of this field, we classify these strategies based on the combination of QD types and detection targets, including traditional QDs or QD micro/nano-spheres-FLISA, and multiple FLISA platforms. In addition, some new sensors based on the QD-FLISA are introduced; this is one of the hot spots in this field. The current focus and future direction of QD-FLISA are also discussed, which provides important guidance for the further development of FLISA.

Establishment of a Ca(II) ion-quantum dots fluorescence signal amplification sensor for high-sensitivity biomarker detection.

Quantum dots (QDs) have been considered as the promising fluorescent labeling material, which is expected to meet the requirement of high-sensitivity detection in clinical diagnostics. Some common metal ions are known to affect the stability and fluorescence properties of QDs, but scarcely any systematic research has been done about their impacts on QD-based bio-detection. By evaluating the effect of Ca2+ metal ions on the properties of aqueous QDs, a new metal ion-QD fluorescence signal amplification sensor (i.e., Ca2+-QD-fluorescence-linked immunosorbent assay, Ca2+-QD-FLISA) has been developed for the detection of inflammatory biomarkers with high sensitivity. Compared with the common QD-FLISA, the detection sensitivity for CRP of Ca2+-QD-FLISA was improved by a 4-fold of magnitude to 0.23 ng/mL, and this assay showed good selectivity, high accuracy, and excellent repeatability. The versatility of the QD-FLISA method were also validated by using different metal ion-QD probes (Ca2+, Mg2+, Ba2+, Fe2+, and Mn2+) to detect CRP, serum amyloid A (SAA), and procalcitonin (PCT). The significant improvement in detection sensitivity was achieved due to the crosslinking of aqueous QDs by Ca2+ ions to enhance fluorescence and at the same time promote antigen-antibody binding efficiency. The present study illustrates the versatility of metal ion-QD-FLISA as a simple and effective method to detect a wide range of biomarkers with high sensitivity and accuracy.

Aminophosphate precursors for the synthesis of near-unity emitting InP quantum dots and their application in liver cancer diagnosis.

InP quantum dots (QDs) are a promising and environment-friendly alternative to Cd-based QDs for in vitro diagnostics and bioimaging applications. However, their poor fluorescence and stability severely limit their biological applications. Herein, we synthesize bright (∼100%) and stable InP-based core/shell QDs by using cost-effective and low-toxic phosphorus source, and then aqueous InP QDs are prepared with quantum yield over 80% by shell engineering. The immunoassay of alpha-fetoprotein can be detected in the widest analytical range of 1-1000 ng ml-1 and the limit of detection of 0.58 ng ml-1 by using those InP QDs-based fluorescent probes, making it the best-performing heavy metal-free detection reported so far, comparable to state-of-the-art Cd-QDs-based probes. Furthermore, the high-quality aqueous InP QDs exhibit excellent performance in specific labeling of liver cancer cells and in vivo tumor-targeted imaging of live mice. Overall, the present work demonstrates the great potential of novel high-quality Cd-free InP QDs in cancer diagnosis and image-guided surgery.

Self-assembly strategy to reduce non-specific adsorption for the development of high sensitivity quantitative immunoassay.

The development of functionalized surfaces with low non-specific adsorption is important for their biomedical applications. To inhibit non-specific adsorption on glass substrate, we designed a novel optical biochip by modifying a layer of dense negatively charged film (SO32-) on its substrate surface via self-assembly. Compared with the untreated glass substrate, it reduced the adsorption by about 300-fold or 400-fold by poly (styrene sulfonic acid) sodium salt (PSS), or meso-tetra (4-sulfonatophenyl) porphine dihydrochloride (TSPP) on individually the modified glass substrate. Considering the effect of fluorescence resonance energy transfer (FRET) between TSPP and the QDs in solution by mixing, a strategy of 2-layer of TSPP followed by 4-layer of PSS was designed to modify the glass for preparing biochips. Under the optimized conditions, the biochip on functionalized glass substrate co-treated with TSPP and PSS realized the sensitive quantitative detection of C-reactive protein (CRP) based on a quantum dot fluorescence immunosorbent assay (QD-FLISA). The limit of detection (LOD) for CRP achieved 0.69 ng/mL with the range of 1-1,000 ng/mL using TSPP and PSS co-treated glass substrate surface, which was respectively about 1.9-fold and 7.5-fold more sensitive to the PSS-modified biochip and the TSPP-modified biochip. This work demonstrated an effective and convenient strategy to obtain biochips with low non-specific adsorption properties on functionalized surfaces, thus providing a new approach for creating ultra-high sensitivity microchannels or microarrays on glass substrates.

Synthesis of Reabsorption-Suppressed Type-II/Type-I ZnSe/CdS/ZnS Core/Shell Quantum Dots and Their Application for Immunosorbent Assay.

We report a phosphine-free one-pot method to synthesize ZnSe/CdS/ZnS core-shell quantum dots (QDs) with composite type-II/type-I structures and consequent reabsorption suppression properties. The as-synthesized QDs possess high efficient red emission (with quantum yield of 82%) and high optical stability. Compared to type-I QDs, the ZnSe/CdS/ZnS QDs show larger Stokes shift and lower reabsorption which can reduce the emission loss and improve the level of fluorescence output. The ZnSe/CdS/ZnS QDs are used as fluorescent labels to exploit their application in fluorescence-linked immunosorbent assay (FLISA) for the first time in the detection of C-reactive protein (CRP) with a limit of detection (LOD) of 0.85 ng/mL, which is more sensitive than that of CdSe/ZnS type-I QDs based FLISA (1.00 ng/mL). The results indicate that the ZnSe/CdS/ZnS type-II/type-I QDs may be good candidates for applications in biomedical information detection.

Functional Connectivity Differences in the Insular Sub-regions in Migraine without Aura: A Resting-State Functional Magnetic Resonance Imaging Study.

Objective: The objective of this study was to investigate resting-state functional connectivity (FC) differences in insular sub-regions during the interictal phase in patients with migraine without aura (MWoA). Methods: Forty-nine MWoA patients (MWoA group) and 48 healthy individuals (healthy control group) were recruited for this study. All of the subjects underwent neurological examination and magnetic resonance imaging (MRI). The MRI data were processed using Brat 1.0 software to obtain a whole-brain FC diagram and using Rest 1.8 software to obtain the FC z-score of the sub-regions of both insulas (six sub-regions on each side). Therefore, there were a total of 12 regions of interest (ROIs) that were used as seed points for the statistical analysis. Results: There was abnormal FC between the insular sub-regions and multiple brain regions in the MWoA patients compared with the healthy control group, and a clear laterality was also observed. In addition, the FC z-score of certain sub-regions was negatively correlated with the disease duration. Conclusion: Different insular sub-regions are functionally associated with different regions of the brain and therefore have different functions. In MWoA, the FC between the insular sub-regions and other brain regions was mostly reduced, while a small amount was increased; additionally, the FC may be ipsilateral with a right-side advantage. Variations in the FC of insular sub-regions can be observed as an important indicator of MWoA.

Different mean thickness implicates involvement of the cortex in migraine.

The results of neuroimaging studies on migraines have shown that the functions and functional connectivity networks of some brain regions are altered in migraine patients, and different brain structure volumes have also been observed in recent years. However, it is still not known whether the mean thickness of the cortex is different in migraine patients.A total of 48 migraine without aura (MWoA) patients in interictal phase and 48 healthy controls were enrolled in this study. All subjects received neurological and magnetic resonance imaging (MRI) examinations. Automatic segmentation processing of high-resolution MRI structure images was performed using FreeSurfer software.The mean cortical thickness of many brain regions in the frontal lobe, temporal lobe, occipital lobe, parietal lobe, and insula in the migraine patient group was significantly decreased compared with that in the healthy control group. The mean cortical thickness of the insula anterior was positively correlated with the duration of the disease course, while the mean cortical thickness of insula superior and insula inferior was negatively correlated with the duration of the disease course.The results showed that MWoA results from a complex interactive reaction involving many brain regions and many brain network systems together. However, it is still not clear whether the difference in the brain structure of migraine patients is the result or the cause of headache, which is a topic that must be better elucidated. Therefore, longitudinal neuroimaging studies on migraine patients with large samples sizes should be performed using more advanced neuroimaging techniques.