A highly sensitive fluorescent immunosensor for sensitive detection of nuclear matrix protein 22 as biomarker for early stage diagnosis of bladder cancer

Abstract

A novel strategy is reported for highly sensitive, rapid, and selective detection of nuclear matrix protein NMP22 using two-color quantum dots based on fluorescence resonance energy transfer (FRET). Quantum dots (QDs) are highly advantageous for biological imaging and analysis, particularly when combined with (FRET) properties of semiconductor quantum dot (QDs) are ideal for biological analysis to improve sensitivity and accuracy. In this FRET system narrowly dispersed green emitting quantum dot CdTe core is used as a donor and labelled by monoclonal (mAb) antibody, while orange emitting quantum dot CdTe/CdS core shell is used as an accepter and labelled by polyclonal (pAb) antibody. The quantum dots are labelled by antibodies using EDC/NHS as crosslinking agent. Bovine serum albumin (BSA) solution was added to block nonspecific binding sites. The fluorescence intensity of QDs accepter decreased linearly with the increasing concentrations of NMP22 from 2-22 pg mL^-1 due to FRET system and fluoroimmunoassay reaction. This method has good regression coefficient (R ² = 0.998) and detection limit was 0.05 pg mL^-1. The proposed FRET-based immunosensor provides a quick, simple and sensitive immunoassay tool for protein detection, and can be considered as a promising approach for clinical applications. The proposed FRET-based immunosensor provides a quick, simple and sensitive immunoassay tool for protein detection, and can be considered as a promising approach for clinical applications.

Fig. 1. Illustration of FRET based detection of NMP22.

Fig. 2. TEM image for (a) CdTe core quantum dot (b) CdTe/CdS core–shell quantum dot and (c) is X-ray diffraction (XRD) for CdTe core and CdTe/CdS core–shell.

Fig. 3. Fluorescence spectra (a) of green-emitting QD_(D) and (b) of orange-emitting QD_(A) absorption spectra (d) of green-emitting QD_(D) and (c) of orange-emitting QD_(A).

Fig. 4. Fluorescence spectra of (a) different ratio (1 : 1, 1 : 2, 1 : 4, 2 : 1 and 4 : 1) μL QD_(D)–mAb mixed QD_(A)–pAb. (b) (a): PL spectra of QD_(D)–mAb, (b): QD_(A)–pAb and (c): QD_(D)–QD_(A) system λ_ex = 340 nm.

Fig. 5. Time resolved of PL intensity of QD_(D) and QD_(D) in presence of acceptor (FRET system).

Fig. 6. (a) Fluorescence spectra of FRET mechanism form 2 pg mL⁻¹ to 22 pg mL⁻¹, (b) calibration curve for detection of NMP22 from 2 pg mL⁻¹ to 22 g mL⁻¹.