Construction Of High-sensitive Optical Sensor Based On CdSe/ZnS Quantum Dot Flurescence Immunoassay

The need for highly accurate and sensitive detection technology are increasingly in the biomedical field,especially for the detection of various biomolecules,including biomarkers,aptamers and proteins.Accurate and rapid acquisition of much biological information from a small or trace amount of sample is the trend for its further development.Among them,how to effectively apply biomarkers to quantitative assays is of great significance for clinical diagnosis.Quantum dots(QDs)as a new type of fluorescent probes are widely used in for biomarkers detection due to their unique optical properties.Biosensors constructed based on the optical properties of QDs use QDs and QDs-biocouples as targets for biomolecular sensors,and the contents of the detected substance are determined by measuring the fluorescence intensity signal.As one kind of QDs optical biosensors,quantum dots-based fluorescence-linked immunosorbent assay(QD-FLISA or QLISA)has achieved quantitative detection of specific proteins and small molecule toxins due to its high sensitivity,accuracy,and high throughput detection and other characteristics.Although more and more research on QD-FLISA has been devoloped,there are still some problems that need to be overcome for use in in vitro diagnotic,such as co-detection of multiple substances,sensitivity,and accuracy.Thus,in this paper,we constructed several kinds of highly sensitive optical biosensors based on different detection substrates by QD-FLISA to achieve high accuracy and sensitivity for quantitative detection of inflammation biomarkers.The specific work carried out is as follows.(1)The simultaneous detection of the inflammation biomarkers by optical biosensor based on dual quantum dots fluorescence immunosorbent assay.We selected C-reactive protein(CRP)and serum amyloid A(SAA)as target inflammation biomarkers,and prepared two-color QDs-probe probes using highly stable red and green aqueous QDs labeled with SAA and CRP antibodies,respectively.After optimizing the detection conditions and sample added sequence,we successfully achieved the simultaneous detection of both SAA and CRP in one reaction mircoplate well.The method provided a relatively wide linear range of 10-1,000 ng/m L for the detection of SAA and CRP,and the limit of detection(LOD)were 2.39 ng/m L and 6.37 ng/m L,respectively.The recoveries of this method ranged from 92.13%to 101.85%with the coefficient of variance(CV)less than 15%.The above results indicate that the method has good stability and accuracy.(2)Based on the optimization of the coating antibodies method to improve the sensitivity of QD-FLISA biosensor.The immobilization of the coating antibodies on the microplate is usually based on non-covalent bonding by intermolecular forces,showing a disordered state,which makes some antibodies unable to bind effectively to the antigen,thus preventing further improvement of the detection sensitivity.We conducted quantitative detection of CRP with QD-FLISA by using silylation reagents(APS,GPS,and MPS)to modify the surface of the substrate of the microplate,which orderly binded coating antibodies through chemical bonds combination methods.The APS-treated microplates were better than the directly coating antibody(non-covalent bonding coating),and the sensitivity of the detection was improved to 0.76 ng/m L.The MPS-treated microplates had the best ability to capture high concentrations of CRP antigens,and the sensitivity of the detection could be improved to 0.65 ng/m L.(3)The construction of a novel optical biosensor based QD-FLISA with glass as substrate.In order to inhibit the adsorption of the QDs probe on the glass substrate,the glass substrate has been treated with polystyrene sulfonic acid sodium(PSS)or meso-Tetra(4-sulfonatophenyl)porphine dihydrochloride(TSPP,sulfonic acid porphyrin)containing negative charge by layer self-assembly(LBL)and spin-coating method,respectively,which inhibited the adsorption of aqueous QDs probes on the glass substrate.After surface-modified glass substrates were coated with CRP antibodies,the quantitative detection of CRP was successfully realized using QD-FLISA.Both the PSS-modified glass substrate chip and the TSPP-modified glass substrate chip had achieved the quantitative detection of CRP wtih the LOD was 1.26 ng/m L and 5.17 ng/m L,respectively.When the glass substrates were co-treated both TSPP and PSS,the LOD for CRP has been improved to 0.69 ng/m L.Besides,the quantitative detection of CRP was performed in the LOD of 1ng/m L using PSS modified glass substrates by spin-coating method.In addition,we used polydimethylsiloxane(PDMS)and glass substrates to prepare the biochip microarrays,and MPS was applied to functionalize the surface modification of the biochip substrate.The quantitative detection of CRP was successfully achieved,and the detection sensitivity could also reach 0.89 ng/m L.(4)The construction of a three-dimensional immunochip sensor for co-detection of inflammation biomarkers with single color quantum dots.Due to the limited adsorption capacity of common substrates(microplates or glass substrates as two-dimensional planar substrates),a 3D structrate of the"groove"substrate has been designed to further improve the detection sensitivity,which had high antibody capture capacity(7 times higher than that of the 2D substrate)and near the"absolute zero"background in immunosaasy.The piezoelectric microarrayer was used to spot samples at different positions of the same reaction well,and a single color QDs probe was used to achieve quantitative detection of the two substances to be measured,and the fluorescence intensity was collected by a self-built fluorescence spectrometer.After optimizing the experimental conditions,we successfully achieved the quantitative detection of CRP andSAA,both with an extended detection range of 0.1-1,000 ng/m L and LOD of 0.11 ng/m L of CRP and 0.16 ng/m L of SAA,respectively,which showed a great improvement in sensitivity compared to the 2D-based detection.The recoveries of this microarray were in the range of 91.1%-114.6%with the CV less than 15%,which indicated that this method the good stability and accuracy.(5)The synthesis of highly stable quantum dot microbeads and its applicability in the new optical sensors are discussed.In order to improve the sensitivity,high quality QDs-MB(size around 200 nm)were prepared by self-assembly of thiolated silicon spheres and oil QDs,which have wider p H stabilization range and better photothermal stability than the silica-coated mono-quantum dot(QDs@Si O₂).The ratio of coupled antibodies was optimized for the MB-QDs,and the optimal ratio of coupled antibodies was to be 1:12.Using this QDs-MB as the fluorescent probe for QD-FLISA,sensitive detection of lower concentrations of CRP was achieved with an LOD of 0.36 ng/m L due to its high fluorescence intensity and more antibody coupling efficiency,which was 2-fold more sensitive than the QD-FLISA method with QDs@Si O₂ as the fluorescent probe.The sensitivity of the quantitative detection of CRP was improved to 0.24 ng/m L using the QDs-MB evaluated on MPS-modified microplate,which was 3-fold higher compared to the QD-FLISA method with QDs@Si O₂ as a fluorescent probe.When the prepared QDs-MB was used in the developed three-dimensional immunochip sensor,the quantitative range of CRP andSAA were extended to 0.05-1,000 ng/m L,and the LOD reached 15pg/m L of CRP and 86 pg/m L of SAA,respectively,achieving ultra-sensitive detection for biomarkers.