Light-Up Aptameric Sensor of Serotonin for Point-of-Care Use.

Serotonin is a vital neurotransmitter for regulating organism functions, and its abnormal level indicates multiple diseases. Aptamer has emerged as an innovative tool for serotonin analysis very recently; however, the current aptameric sensing platform lacks design flexibility and portability. Here, we introduce a light-up aptameric sensor using designer DNA molecules with tunable affinity and dynamic response and achieve mobile phone-based detection for point-of-care use. We develop a type of allosteric DNA sensor through flanking the serotonin recognition domain with split fluorogenic sequences, where both linker lengths and split sites of the aptamer affect its function. In addition, we design a series of molecular constructs that contain nucleotide mutations and systematically investigate the structure folding and ligand binding of the aptameric molecules. The results show distinct effects of variant mutation sites on conformation change and sensing responses. Notably, the variable aptameric molecules allow affinity and dynamic response regulation, which are adaptable to diverse sensing applications that require different threshold levels. Furthermore, we demonstrate a simple surface-based assay that can use smartphone imaging to visualize results for diagnosis. In a portable and simple manner, highly sensitive and selective serotonin assay is achieved in different biofluids, with detection limits in the low nanomolar range. This study offers an alternative approach for serotonin assay using engineered aptameric molecular probes. We expect that the practical utility may make the method promising in resource-limited settings.

SERS-fluorescence dual-mode nanoprobe for the detection and imaging of Bax mRNA during apoptosis.

A dual-mode nanoprobe was constructed to detect Bax messenger RNA (mRNA), consisting of gold nanotriangles (AuNTs), a Cy5-modified recognition sequence, and a thiol-modified DNA sequence. Bax mRNA is one of the key pro-apoptotic factors in the apoptosis pathway. Raman enhancement and fluorescence quenching of the signal group Cy5 were performed using AuNTs as substrates. The thiol-modified nucleic acid chain is partially complementary to the Cy5-modified nucleic acid chain to form a double strand and is linked to the AuNTs by the Au-S bond. When Bax mRNA is present, the Cy5-modified strand specifically binds to it to form a more stable duplex, making Cy5 far away from AuNTs, and SERS signal is weakened while fluorescence signal is enhanced. The nanoprobe can be used for the quantitative detection of Bax mRNA in vitro. Combined with the high sensitivity of SERS and the visualization of fluorescence, this method has good specificity and can be used for in situ imaging and dynamic monitoring of Bax mRNA during deoxynivalenol (DON) toxin-induced apoptosis of HepG2 cells. DON plays a pathogenic role mainly by inducing cell apoptosis. The results confirmed that the proposed dual-mode nanoprobe has good versatility in various human cell lines.

Fluorescence-Raman dual-mode quantitative detection and imaging of small-molecule thiols in cell apoptosis with DNA-modified gold nanoflowers.

The monitoring of small-molecule thiols (especially glutathione) has attracted widespread attention due to their involvement in numerous physiological processes in living organisms and cells. In this work, a dual-mode nanosensor was designed to detect small-molecule thiols, which is based on the "on-off" switch of fluorescence resonance energy transfer (FRET) and surface-enhanced Raman scattering (SERS). Briefly, DNA was modified by Cy5 (signal probe) and disulfide bonds (recognition element). Gold nanoflowers (AuNFs) were used as the fluorescence-quenching and SERS-enhancing substrate. However, small-molecule thiols can cleave disulfide bonds and release short Cy5-labeled chains, causing the recovery of the fluorescence signal and a decrease of the SERS signal. The nanosensor showed a sensitive response to small-molecule thiols represented by GSH, with a linear range of 0.01-3 mM and a detection limit of 913 nM. In addition, it competed with other related biological interferences and presented good stability and better selectivity towards small-molecule thiols. Most importantly, the developed nanosensor had been successfully applied to in situ imaging and quantitative monitoring of the concentration of small-molecule thiols which changed during T-2 toxin-induced apoptosis in HeLa cells. Meanwhile, nanosensors are also versatile with their potential applications and can be easily extended to the detection and imaging of other human cell lines. The proposed method combines the dual advantages of fluorescence and SERS, which has broad prospects for in situ studies of physiological processes involving small-molecule thiols in biological systems.