| Quantum dots(QDs)have many excellent fluorescence characteristics such as high fluorescence efficiency,high stability,large Stockes shift,narrow and symmetrical emission spectrum,single-wavelength multicolor excitation,and therefore it can be used as a new fluorescent labeling material in combination with traditional ELISA.Construction of a new type of quantum dot fluorescent immunosorbent assay(QD-FLISA,QLISA),which is expected to overcome the disadvantages of tedious operation and enzyme activity and substrate stability in ELISA.The QLISA methoed can achieve high-sensitivity,multi-index detection without color development operation,and so it is convenient and fast.Moreover,it can obtain stable detection signal under the multiple excitation in a long time.However,as a novel detection method,there are still some problems in its actual application,such as sensitivity and accuracy have not yet meet the clinical application standards.When testing clinicall samples,samples of human blood and other body fluids are complicated,and some common metal ions are inevitable.In previous studies,it was found that some metal ions have fluorescence quenching on quantum dots,and the metal ion sensors were developed to achieve highly sensitive quantitative detection of metal ions according to the quenching principle.How these metal ions affect the fluorescence signal in QLISA will further affect the accuracy and reliability of the method.Therefore,it is of great significance to explore the influence of these metal ions on the aqueous phase quantum dots and their QLISA detection method,and there are no relevent research work so far.In this study,the effect of different metal ions on water-phase quantum dots and the application of this effect in QLISA were investigated,and a new type of metal ion enhanced-fluorescent immunosorbent assay(Metal ion-QLISA)have been developed.(1)The effect of different metal ions on the aqueous quantum dots.According to the traditional synthesis methods of water-phase quantum dots including ligand exchange method,silica coating method and polymer coating method,the hydrophobic CdSe/ZnS QDs were transfered into the water,and-3-mercaptopropionic acid-modified quantum dots(QDs@MPA),silica-coated quantum dots(QDs@SiO2),and polymer-coated quantum dots(QDs@PMAH)were obtained.Subsequently,nine metal ions including Ca2+,Mg2+,Ba2+,Fe2+,Mn2+,Co2+,Ni2+,Cu2+,and Ag+were respectively added to the three kinds of hydrophilic quantum dots,and it was found that Ca2+,Mg2+,and Ba2+would not quench the the fluorescence of three kinds of hydrophilic quantum dots;When a certain amount of Mn2+,Fe2+,Co2+,and Ni2+are added,the fluorescence of three kinds of water-phase quantum dots are quenched.The addition of Cu2+and Ag+can completely quench the fluorescence of three kinds of hydrophilic quantum dots.The effects of Ca2+,Mg2+,Ba2+,Fe2+,and Cu2+on the hydrodynamic size of three kinds of hydrophilic quantum dots were explored.As the amounts of ions added gradually increased,the hydrated particle size of the hydrophilic quantum dots gradually increased until agglomeration and precipitation occurred.Then it was found that the fluorescence complexes formed could maintain the hydrodynamic size and potential stability in the range of 25?-45?,when a certain amount of five kinds of metal ions(Ca2+,Mg2+,Ba2+,Fe2+,Cu2+)were added to the three kinds of water-phase quantum dots,respectively.(2)The influence of metal ions on the QLISA based on quantum dots.A certain amount of metal ions was added to the probe(QD-mAb)of QLISA to prepare metal ion-QD-mAb probe,and optimize the optimal concentration of different metal ions.The reaction system of the Metal-ion-QLISA method was optimized and the phosphate buffered saline(PBS)contained calf serum was determined as the optimal reaction buffer.Then the Ca2+-QLISA was selected to optimize the reaction time when different kinds of quantum dot probes were used.Finally,the optimal conditions were used to quantitatively detect C-reacetive protein(CRP)by various Metal ion-QLISA methods.The experimental results showed that the fluorescence signals of 500 ng/mL CRP was detected with Mg2+-QLISA,Ca2+-QLISA and Fe2+-QLISA with QDs@SiO2 as fluorescent labels were 3.2 times,3.9 times and 3.5 times of the original QLISA,respectively.Correspondingthe limit of detection of Mg2+-QLISA,Ca2+-QLISA and Fe2+-QLISA was 0.17 ng/mL,0.24 ng/mL,and 0.61 ng/mL,and the sensitivity is 5.6 times,4.2 times and 1.6 times of the original QLISA,respectively.The fluorescence signal of 500 ng/mL CRP with Ba2+-QLISA and Mn2+-QLISA was 2.8 times and 1.3 times that of the original QLISA,but the sensitivity was not improved.The fluorescence signal of Metal ion-QLISA using QDs@PMAH and QDs@MPA as fluorescent labels to detect 500 ng/mL CRP is also stronger than that of QLISA.Fe3+ and Al3+as trivalent metal ions were introduced into traditional QLISA to detect CRP,it was found that the fluorescent signal was also effectively improved.The concentration of these metal ions added in the Metal ion-QLISA is much greater than the concentration of metal ions in body fluids,that is,the metal ions in body fluids will not affect the QLISA results.And the appropriate concentration of metal ions(Mg2+,Ca2+,Fe2+,Ba2+,Mn2+,Fe3+,Al3+)not only does not quench the fluorescence of quantum dots,but also improves the signal of QLISA detection,especially the addition of Mg2+,Ca2+ and Fe2+ can improve the sensitivity of QLISA detection.In addition,inflammatory factors(serum amyloid A and procalcitonin)were detected with Metal ion-QLISA,and the fluorescence signals were effectively improved.The results proved that this novel Metal ion-QLISA with the addition of suitable concentration of metal ions amplifies fluorescence signal of traditional QLISA,and this method is simple to operate and has good versatility. |
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