Affinity binding-mediated fluorometric protein assay based on the use of target-triggered DNA assembling probes and aptamers labelled with upconversion nanoparticles: application to the determination of platelet derived growth factor-BB.

The target-triggered DNA assembling probe is presented for highly selective protein detection. Target-triggered DNA assembling is used in an amplification strategy based on affinity binding for identification and determination of proteins in general. Specifically, it was applied to the platelet derived growth factor-BB (PDGF-BB). A hairpin DNA (H-DNA) probe was designed containing (a) an aptamer domain for protein recognition and (b) a blocked DNAzyme domain for DNAzyme cleavage. An assistant DNA (A-DNA) probe containing aptamer and complementary domains was also employed to recognize protein and to induce DNA assembly. Once H-DNA and A-DNA recognize the same protein, H-DNA and A-DNA are in close proximity to each other. This induces DNA assembling for protein-triggered complex (Protein-Complex) with free DNAzyme domains. The free DNAzymes trigger the circular cleavage of molecular beacons for amplified signals. The assay is performed by fluorometry at an excitation wavelength of 980 nm and by collecting fluorescence at 545 nm. The platelet derived growth factor-BB (PDGF-BB) was accurately identified and selectively determined by this assay with a 22 pM detection limit (using the 3σ criterion). The responses for PDGF-BB is nearly 6-fold higher than for PDGF-AB, and 16-fold higher than PDGF-AA. This upconversion assay avoids any interference by the autofluorescence of biological fluids. Graphical abstractSchematic representation of the principle of the target-triggered DNA assembling probes mediated amplification strategy based on affinity binding for PDGF-BB. The UCNP probe is used for the quantitation of PDGF-BB with high selectivity.

Influence of drugs on the prospective diagnostic method for coronary heart disease with urine.

The morbidity of coronary heart disease (CHD) with high risks has been rising in recent years. A novel and noninvasive method based on surface-enhanced Raman spectroscopy (SERS) was proposed by Yang et al. (Analyst 143: 2235, 2018) to prospectively diagnose the arterial blockage by detecting platelet-derived growth factor-BB (PDGF-BB) in urine. Clinically, anti-platelet drugs (such as aspirin, statins and clopidogrel) are often used for ordinary CHD patients or patients with percutaneous coronary intervention (PCI). Therefore, whether the previous developed method can be applied to the CHD patients on long-term medication (more than 6 months) or post-PCI patients was investigated here. Firstly, urine samples of 13 CHD patients on long-term medication (aspirin, rosuvastatin, clopidogrel bisulfate) and 13 post-PCI patients were measured by the proposed method. Clinical data of coronary angiography results provided by Xin Hua Hospital and Yangpu District Central Hospital Antu Branch revealed that these 26 patients were with serious arterial blockage, however, characteristic Raman peak at 1509 cm-1 attributed to PDGF-BB was not observed in the SERS spectra of these 26 patients. In addition, an eight-day follow-up investigation was performed on a CHD patient with PCI three years ago and on long-term medication. It was found that the Raman peak at 1509 cm-1 could be only observed in the third and fourth day after suspending the drugs. Furthermore, SERS spectra of mixed solutions of PDGF-BB and aspirin, rosuvastatin, mixed solutions of these two drugs and clopidogrel bisulfate were analyzed. The Raman peak at 1509 cm-1 was not found in all these spectra, it indicated that all the three kinds of drugs could influence on the SERS signal of PDGF-BB. Therefore, the previous developed method is not suitable for CHD patients on long-term medication and post-PCI patients.