Chen, Piaopiao, et al. Analyst 144.4 (2019): 1147-1152.
Ascorbic acid (AA) and alkaline phosphatase (ALP) are crucial coenzymes and enzymes involved in various biological metabolic reactions, and abnormal levels of these substrates are associated with multiple diseases. In this study, a novel, simple fluorescence strategy using CdTe quantum dots (QD) as an effective signal indicator was developed for sensing AA and ALP. This method is primarily based on the selective fluorescence quenching reaction between Ag+ and CdTe QDs, rather than silver nanoparticles (Ag NPs); Ag+ can be reduced by AA to form Ag NPs. Moreover, this strategy further enables the determination of ALP by employing AA as an intermediary, as ALP can hydrolyze L-ascorbic acid-2-phosphate (AAP), thus generating AA.
ALP Detection by Adding AAP
Sensitive fluorescence detection of ALP begins with the addition of 50 μL AAP (1 mM) and 50 μL ALP (at various concentrations) into MOPS buffer (100 μL). The resultant solution mixture is then incubated at 37 °C for 30 minutes. Next, 50 μL of Ag+ solution (5 μM) is added to the mixture and incubated at room temperature in the dark for 20 minutes to form Ag NPs. Subsequently, 10 μL of CdTe QDs (a 10-fold dilution of the original CdTe QDs solution) is added to the above mixture and maintained at room temperature in the dark for an additional 10 minutes. Finally, the resultant solution is diluted to 1 mL, and fluorescence measurements are immediately conducted (with an excitation wavelength of 365 nm). All measurements are performed at least three times.
Wang, Mo, et al. Biosensors and Bioelectronics 53 (2014): 232-237.
A photoelectrochemical (PEC) biosensor for the sensitive and specific detection of microRNA was fabricated by integrating Bi2S3 nanorods with enzyme-mediated signal amplification. The l-ascorbic acid 2-phosphate trisodium salt (AAP) was catalyzed by alkaline phosphatase to generate ascorbic acid (AA) in situ, which served as an electron donor. Consequently, the PEC response was positively correlated with the AA electron donor concentration, enabling successful implementation of a signal-on protocol for microRNA detection.
Detection Strategy: Bi2S3 nanorods and AuNPs were successively immobilized on the ITO surface via physical adsorption. Then, a hairpin-structured thio-modified probe DNA was assembled onto the AuNPs/Bi2S3/ITO surface through Au-S bonding. By hybridizing with microRNA, the stem-loop structure of the probe DNA was open. Biotin-labeled DNA was then hybridized with the unhybridized fragment at the 3' end of the probe DNA to immobilize biotin on the electrode. Subsequently, immune gold-labeled streptavidin (SA-AuNPs) was captured via the specific interaction between biotin and streptavidin. Finally, biotin-labeled alkaline phosphatase (biotin-ALP) was introduced into the PEC system to catalyze the hydrolysis of l-ascorbic acid 2-phosphate trisodium salt (AAP) to generate AA. It is evident that immobilization of biotin-ALP depended on the hybridization of microRNA with the probe DNA to open its hairpin structure. Importantly, an increase in microRNA concentration led to a higher loading of SA-AuNPs, thereby increasing the immobilization amount of biotin-ALP, which enhanced the PEC response due to the augmented AA concentration from the catalytic reaction. Utilizing the high selectivity of the hairpin-structured DNA probe, signal amplification by immune gold-labeled streptavidin, and the light-active material from Bi2S3 nanorods, the proposed system could achieve sensitive and selective detection of microRNA.
Liu, Hua, et al.Nanoscale 12.18 (2020): 10390-10398.
Detecting enzyme activity can provide valuable insights for clinical diagnostics. Herein, gold nanobipyramid@MnO2 nanoparticles (AMNS) are synthesized for the first time as surface-enhanced Raman spectroscopy (SERS) substrates, and an "on" SERS strategy is designed to detect enzyme activity without the need for complex SERS nano-label preparation. In the presence of alkaline phosphatase (ALP), sodium 2-phosphate-L-ascorbate (AAP) can be hydrolyzed into ascorbic acid (AA), which can etch the AMNS shell by reducing MnO2 to Mn2+. The breakdown of the MnO2 shell caused by the enhancement of the electromagnetic field of AMNS can lead to a significant increase in the Raman intensity of molecules adsorbed on the nanobipyramids' surface (i.e., crystal violet, CV). Therefore, ALP activity can be accurately quantified based on the MnO2 shell thickness-dependent Raman signal output of CV.
ALP Assay
To assay ALP, add 20 μL of the solution containing different amounts of ALP in a series of 200 μL test tubes. Then, add AMNS (40 μL, 2 OD), AAP (90 μM, 20 μL), and CV (30 μM, 20 μL). Next, dilute the solution to 200 μL using Tris-HCl buffer (10 mM, pH 8.6) and mix thoroughly. Incubate the mixture at 37 °C for 40 minutes, then use a capillary to extract the solution for Raman measurement.
Wang, Wanlu, and Xianwen Kan. Langmuir (2024).
The COVID-19 pandemic caused by the SARS-CoV-2 virus has rapidly spread worldwide, underscoring the need and driving efforts to explore effective diagnostic tests for sensitive detection of the SARS-CoV-2 virus. This work developed an aggregation-induced electrochemiluminescence (AIECL) sensor for ultrasensitive detection of the SARS-CoV-2 nucleocapsid (N) protein. Tetraphenylethylene (TPE) doped in a zeolitic imidazolate framework-90 (TPE-ZIF-90) exhibited efficient aggregation-induced emission (AIE), endowing TPE-ZIF-90 with high ECL intensity. By introducing an alkaline phosphatase (ALP)-modified gold nanoparticle (AuNP)-decorated zinc oxide (ZnO) nanoflower (ALP/Au-ZnO) composite material after immunorecognition capture of the SARS-CoV-2 N protein on the sensing platform, L-ascorbic acid 2-phosphate trisodium salt (AA2P) was catalyzed to generate PO43- and ascorbic acid (AA). Based on a multi-quenching ECL signal strategy, including resonance energy transfer (RET) between TPE-ZIF-90 and Au-ZnO, disassembly of TPE-ZIF-90 triggered by the strong complexation between PO43- and Zn2+, and in situ generated AuNPs from the TPE-ZIF-90 and AA reduction reaction, the constructed AIECL sensor achieved highly sensitive detection of the SARS-CoV-2 N protein with a detection limit of 0.52 fg/mL.
Jing, Yuanxiang, et al. Chemical Engineering Journal 487 (2024): 150561.
A self-healing antibacterial hydrogel dressing with suitable mechanical properties, excellent antioxidant capacity, conductivity, dual ROS/glucose-responsive drug release capability, and biocompatibility was synthesized based on dual dynamic bonds of boronate ester and hydrogen bond. This hydrogel is intended for use in repairing Type I diabetes wounds.
Preparation of P-LP-PMX-CA-L@E Hydrogel
First, 2% LG-PBA and 10% PVA aqueous solutions were prepared separately by mass fraction. Next, a mixture containing 5 mg of polydopamine-modified MXene (PMX), 10 mg of chlorogenic acid (CA), 50 μg of L-ascorbic acid-2-phosphate trisodium salt (L), and 100 μg of exosomes derived from ADSCs (E) was added to the 2% LG-PBA solution. Finally, this solution was directly mixed and stirred with a 10% PVA solution (w/w ratio = 2:3) to obtain the P-LP-PMX-CA-L@E hydrogel. Control groups of hydrogel were also prepared, including P-LP hydrogel (without polydopamine-modified MXene, chlorogenic acid, L-ascorbic acid-2-phosphate trisodium salt, and ADSC-derived exosomes), P-LP-PMX hydrogel (without chlorogenic acid, L-ascorbic acid-2-phosphate trisodium salt, and ADSC-derived exosomes), and P-LP-PMX-CA hydrogel (without L-ascorbic acid-2-phosphate trisodium salt and ADSC-derived exosomes).
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