An alkaline phosphatase-induced immunosensor for SARS-CoV-2 N protein and cardiac troponin I based on the in situ fluorogenic self-assembly between N-heterocyclic boronic acids and alizarin red S.

Alkaline phosphatase (ALP)-induced in situ fluorescent immunosensor is less investigated and reported. Herein, a high-performance ALP-labeled in situ fluorescent immunoassay platform was constructed. The developed platform was based on a fluorogenic self-assembly reaction between pyridineboronic acid (PyB(OH)2) and alizarin red S (ARS). We first used density functional theory (DFT) to theoretically calculate the changes of Gibbs free energy of the used chemicals before and after the combination and simulated the electrostatic potential on its' surfaces. The free ARS and PyB(OH)2 exist alone, neither emits no fluorescence. However, the ARS/PyB(OH)2 complex emits strong fluorescence, which could be effectively quenched by PPi based on the stronger affinity between PPi and PyB(OH)2 than that of ARS and PyB(OH)2. PyB(OH)2 coordinated with ARS again in the presence of ALP due to the ALP-catalyzed hydrolysis of PPi, and correspondingly, the fluorescence was restored. We chose cTnI and SARS-CoV-2 N protein as the model antigen to construct ALP-induced immunosensor, which exhibited a wide dynamic range of 0-175 ng/mL for cTnI and SARS-CoV-2 N protein with a low limit of detection (LOD) of 0.03 ng/mL and 0.17 ng/mL, respectively. Moreover, the proposed immunosensor was used to evaluate cTnI and SARS-CoV-2 N protein level in serum with satisfactory results. Consequently, the method laid the foundation for developing novel fluorescence-based ALP-labeled ELISA technologies in the early diagnosis of diseases.

Simple construction of a two-component fluorescent sensor for turn-on detection of Hg2+ in human serum.

The simply constructed fluorescent sensor with inexpensive reagents and low toxicity has attracted increasing attention contributing to its practical application. However, the common construction methods usually required a few building blocks and complex procedures, which is inconvenient for their further application. Herein, a simply constructed fluorescent Hg2+ sensor has been developed based on the intrinsic fluorescence quenching power of G-quadruplex. Two components, AGRO 100 and AMT, were used to construct the sensor. AMT was selected as the fluorescent probe because of its distinct merits. The free AMT emits strongly. However, the fluorescence of AMT could be quenched by G-quadruplex DNA. Additionally, AMT is less toxic and inexpensive. AGRO 100 acts as both the quencher and the capture sequence because it consists of G-rich sequences and T-T mismatched base pairs. The fluorescence of AMT could be quenched by the formed G-quadruplex structure of AGRO 100 in the presence of K+. In the presence of Hg2+, G-quadruplex structure of AGRO 100 was switched to hairpin DNA structure because T-T mismatched base pairs in AGRO 100 could specifically recognize and capture Hg2+ with high affinity. Thus, AMT was released and the fluorescence of AMT was recovered. The developed sensing system was successfully applied to detect Hg2+ in human serum with good recovery and reproducibility.

A label-free oligonucleotide based thioflavin-T fluorescent switch for Ag+ detection with low background emission.

A novel fluorescent Ag(+) sensor was developed based on the label-free silver (I) specific oligonucleotide (SSO) and Thioflavine T (ThT) monomer-excimer switch. C-rich SSO which contain C-C mismatched base pairs can selectively bind to Ag(+) ions and the formed duplexes which constructed by C-Ag(+)-C structure are thermally stabilized without largely altering the double helical structure. ThT give very weak fluorescent in bulk solution and/or in the presence of SSO. However ThT shows high fluorescence in the presence of SSO and Ag(+) at the same time mainly because ThT excimer, which has the high quantum yield, formed and stabilized in the minor or major groove. Based on the discovery, we developed the novel Ag(+) sensor. Under the optimum condition, the selectivity of this system for Ag(+) over other metal ions in aqueous solution is remarkably high, and Ag(+) can be quantified over the dynamic range of 30-450 nM, with a limit of detection of ~16 nM and a linear correlation coefficient of 0.995.

A colorimetric and ratiometric photometric sequential assay for ascorbic acid and alkaline phosphatase in serum based on valence states modulation.

The photometric method is widely used in real clinical tests due to its simple operation, low cost and convenient. Many of the reported colorimetric ALP assays so far are non- ratiometric because the detection was based on changes in absorbance at a single wavelength. The development of novel colorimetric and ratiometric assay is of importance for quantitatively measuring target with high accuracy. The challenge in the design of ratiometric photometric assay is that the chromophore must have a significant spectral shift before and after binding to the target. Here, we report a colorimetric and ratiometric photometric sequential assay of AA and ALP based on the complexation between ARS and Cu2+ and redox reaction between AA and Cu2+. The absorption band of ARS centered at 425 nm (yellow color), which could be shifted to 510 nm (red color) upon Cu2+ binding. However, as far as we know, this classic color reaction has not been used to develop a ratiometric photometric method to sequentially detect AA and ALP, although photometric methods based on the regulation of other color reagents with oxidizing metal ions have been reported. The proposed sensing system shows a limit of detection for ALP at 0.24 U L-1 and could be applied for detecting ALP in newborn calf serum. The established sensing system makes a useful contribution to the detection of ALP in complex clinical samples.

Oligonucleotide-based label-free Hg2+ assay with a monomer-excimer fluorescence switch.

A novel fluorescent Hg(2+) sensor was developed based on the T-Hg(2+)-T structure and a thioflavine T monomer-excimer fluorescent switch. Under optimum conditions, the selectivity is remarkably high, and Hg(2+) can be quantified over the dynamic range of 0.1 to 1.2 µM, with a limit of detection (LOD) of ~20 nM and a linear correlation coefficient of 0.995.

Design of a dual-signaling sensing system for fluorescent ratiometric detection of Al3+ ion based on the inner-filter effect.

A dual-signal sensing system based on the inner-filter effect (IFE) was demonstrated, in which the combination of two signaling mechanisms allows metal binding to turn on two fluorescence emission bands, independently. A proof-of-concept fluorescent ratiometric assay for Al(3+) in pure aqueous solution is presented. The proposed assay is based on the Al(3+)-induced color and fluorescence changes of Alizarin red S (ARS) and IFE between ARS and meso-tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP). In the absence of Al(3+), the absorption spectrum of the ARS in 0.2 M HAc-NaAc buffer (pH 5.5) has a strong peak at 420 nm, significantly overlapping with the excitation of TMPyP. ARS is expected to be capable of functioning as a powerful absorber to tune the emission of TMPyP on account of the spectral overlap. Binding of Al(3+) with ARS forms a fluorometric ARS/Al(3+) complex and shifts the maximum absorbance from 420 nm to 480 nm, which overlaps negligibly with the excitation of TMPyP and turns on the proper emission spectrum for TMPyP. Under the optimum conditions, The fluorescence intensity ratio, F(585)/F(651), responds to Al(3+) over a dynamic range of 0.1-1.5 µM, with a limit of detection of 40 nM, where F(585) and F(651) are the fluorescence intensity at 585 nm and 651 nm in the absence or presence of Al(3+), respectively. Further application in Al(3+)-spiked water samples suggested a recovery between 95 and 108%. The fluorescence response is highly selective for Al(3+) over other metal ions with the addition of thiourea as the masking agent.

An improved structure-switch aptamer-based fluorescent Pb2+ biosensor utilizing the binding induced quenching of AMT to G-quadruplex.

An improved aptamer-based fluorescent Pb2+ biosensor utilizing the binding induced quenching of AMT to G-quadruplex has been rationally designed with a LOD of 3.6 nM. The utility of the developed biosensor was demonstrated by the successful detection of Pb2+ in real complex clinical samples with satisfactory recovery and good reproducibility.

Design of a Fluorescence Turn-on and Label-free Aptasensor Using the Intrinsic Quenching Power of G-Quadruplex to AMT.

A novel fluorescent aptasensor based on the G-quadruplex induced fluorescent quenching of psoralen and the competitive interactions between 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), adenosine triphosphate (ATP) and G-rich DNA functionalized split ATP aptamer was proposed. The binding of ATP to the G-rich DNA functionalized split aptamer induced a significant enhancement in fluorescence emission intensity while undergoing excitation at 340 nm. Under the optimal conditions, the developed aptasensor showed high selectivity and good accuracy for detecting ATP. The practicality of the proposed aptasensor has been confirmed by successfully analyzing ATP in spiked human blood serum samples with satisfactory results. As far as we know, this is the first time that the intrinsic quenching ability of G-quadruplex was applied to simply construct a fluorescence turn-on and label-free aptasensor. On account of the superiority of the simplicity of the design strategy, more work is expected in the future to develop a variety of novel sensors for other important analytes using the quenching capability of G-quadruplex through reasonable designs.

Design of a nanoswitch for sequentially multi-species assay based on competitive interaction between DNA-templated fluorescent copper nanoparticles, Cr3+ and pyrophosphate and ALP.

The present work constructs a sequentially triggered nanoswitch (STN) for sequential detection of Cr3+, P2O74- (PPi) and alkaline phosphatase (ALP) depending on polythymine (T40) templated fluorescent Cu nanoparticles (Cu NPs). A significant phenomenon is that Cr3+ can only causing 5% QE of fluorescent Cu NPs synthesized by lower than 500 µM Cu2+, but the fluorescence of the Cu NPs synthesized by more than 500 µM Cu 2+ can be quenched up to 90% QE by the same concentration of Cr3+. Then the quenched fluorescence of CuNP-Cr3+ complex provides a sensing platform for PPi due to the strong binding between Cr3+ and PPi, resulting in dissociation of Cr3+ from the surface of Cu NPs and the recovery of fluorescence emission. Further ALP hydrolysis of PPi disrupts Cr3+-PPi assemble and Cr3+ is released to interact with Cu NPs, which induces fluorescence quenching again. Thus, sequentially detection of Cr3+ (LOD, 0.03 µM), PPi (LOD, 0.005 µM) and ALP (LOD, 0.125 mU/mL) was successfully implemented with high sensitivity and selectivity. The sensor is also successfully used for Cr3+, PPi and ALP assays in the human serum. Additionally, the sensitive "on-off-on-off" sensing behavior of the Cu NPs allow three chemical inputs (Cr3+, PPi and ALP) to construct a logic gate.

Novel strategy to improve the sensing performances of split ATP aptamer based fluorescent indicator displacement assay through enhanced molecular recognition.

Split aptamer strategy was often used to improve the sensitivity of aptasensor. However, traditional split aptamer strategy can not be directly used to improve the label-free aptamer based Thioflavin T (ThT) displacement assay for ATP because the split ATP aptamer display much lower enhancement effects on the fluorescence of ThT than intact aptamer. In order to address this issue, this is the first report using G-rich DNA sequence to enhance the affinity of the two split ATP aptamer halves to ThT and offer lower limit of detection (LOD), wider linear range and higher selectivity through the enhanced molecular recognition. Compared to the intact aptamer/ThT complex, the ensemble of two G-rich split ATP aptamer fragments/ThT are higher fluorescent. Consequently, G-rich sequences would improve the fluorescent signal and thus the sensing performance of the proposed assay. In the optimized conditions, the LOD of the proposed fluorescent ATP aptasensor is 2 nM, which is lower than the reported ThT/ATP aptamer based methods. Additionally, our aptasensor has a wider dynamic linear range (0.1 µM - 120 µM) and higher selectivity. The proposed aptasensor has been successfully applied to detect ATP in 15% human serum. More importantly, the current study not only provides a novel method for ATP assay but also presents a way to construct a label-free split aptamer based fluorescent sensor for other species where aptamer can be generated.

Development of luminescent nanoswitch for sensing of alkaline phosphatase in human serum based onAl3+-PPi interaction and Cu NCs with AIE properties.

As an important biomarker, alkaline phosphatase (ALP) is one of the most commonly assayed enzymes in clinical practice. Here a novel turn-on fluorescent nanoswitch for ALP assay was suggested. The nanoswitch was easily constructed via two high-affinity ligands between GSH and Al3+ and PPi and Al3+ based on the difference in the affinity. The primary fluorescence of as-prepared GSH capped Cu nanoclusters (NCs) turned on first upon the Al3+ addition due to the Al3+ induced aggregation induced emission (AIE) enhancement based on the high affinity of GSH and Al3+. The presence of PPi then made Al3+ desorb from the surface of GSH capped Cu NCs due to the higher affinity of PPi and Al3+. As a result, the fluorescence of the Cu NCs was quenched. ALP could hydrolyze PPi into phosphate, destroying the PPi-Al3+ complex and releasing Al3+. Thus, the Al3+ binds to GSH again and the fluorescence was restored. The nanoswitch was demonstrated to be sensitive and selective for ALP assay and was successfully used for the ALP assay in the human serum.

Fluorescence turn-on and colorimetric dual readout assay of glutathione over cysteine based on the fluorescence inner-filter effect of oxidized TMB on TMPyP.

Quantitative fluorescence turn-on and colorimetric detection of glutathione (GSH) with rapid speed, low cost have attained much attention. Herein, we developed a sensitive fluorescence turn-on and colorimetric sensor for GSH based on the inner-filter effect (IFE), which is the first time to select oxTMB and TMPyP as the IFE absorber and fluorophore pair, respectively. The absorption band of oxTMB matches well with the emission band of TMPyP in the IFE-based fluorescent assay. In the absence of GSH, the absorption peak of oxTMB at 652nm significantly overlaps with the emission of TMPyP, resulting in the efficient IFE and inhibition of the fluorescence of TMPyP. In the presence of GSH, the absorption intensity at 652nm decreases, generating the recovery of the fluorescence of TMPyP. Therefore, this approach is demonstrated to be a novel candidate for detection of GSH, with high sensitivity and selectivity. The linear dynamic range for the concentrations of GSH is between 0.1µM to 20µM along with a limit of detection (LOD) of about 30nM (calculated LOD as 3σ/slope). Finally, this novel sensor was successfully applied for GSH detection in fetal calf serum, and satisfactory recovery was achieved.

A label-free fluorescent molecular switch for a DNA hybridization assay utilizing a G-quadruplex-selective auramine O.

A sensitive and selective assay of DNA is developed by utilizing a signal transduction strategy with the rational redesign of the hairpin structured G-quadruplex molecular switch (G4-MS) assembled using auramine O (AO). By monitoring the changes of the fluorescent signal, we could identify and further quantitatively determine the target DNA in the samples.