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DNA structures with the potential to concurrently recruit multiple ligands are promising in pharmaceutical and sensing applications when concentrated in a local environment. Herein, we found that human telomeric G-quadruplex (htG4) structures with a junction can selectively aggregate a natural ligand of tetrahydropalmatine (THP) into AIEgens. The htG4 monomer favors formation of a THP dimer emitting at ∼525 nm. In addition, only a hybrid htG4 folding supports formation of the emissive THP dimer. However, overhanging a duplex beyond the 5′ end of the hybrid htG4 structure preferentially forms THP J-aggregates with member molecularity being more than two. It is demonstrated that the junction between the duplex and the hybrid htG4 structure is responsible for formation of the THP J-aggregates, as confirmed by the fact that the pairing state of the junction affects the molecularity of the J-aggregates. Nevertheless, such J-aggregates cannot be grown at the junction of two tandem htG4s. Therefore, G4-initiated ligand aggregation (GILA) for natural compounds provides a new way to design pharmaceuticals and sensors with a high local concentration at the site of interest.

Ergothioneine (ERG) is a natural sulfur-containing amino acid found in many organisms, including humans. It accumulates at high concentrations in red blood cells and is distributed to various organs, including the brain. ERG has numerous health benefits and antioxidant capabilities, and it has been linked to various human physiological processes, such as anti-inflammatory, neuroprotective, and anti-aging effects. Accurate, rapid, and cost-effective quantification of ERG levels in human biofluids is crucial for understanding its role in oxidative stress-related diseases. Surface-enhanced Raman scattering (SERS) is an effective approach for measuring compounds at concentrations similar to those at which ERG is present in serum. However, while SERS has been used to characterize or detect ERG, quantification has not yet been achieved due to the variability in the signal enhancement that can arise during sample preparation and analysis. This study introduces a highly efficient and reliable technique for quickly (20 min is typical per sample) measuring ERG levels in human serum using SERS. This employs an internal standard highly specific for ERG which resulted in limit of quantification values of 0.71 μM. To validate this approach, we analysed real human serum with unknown ERG levels as a blind test set and primary reference levels of ERG were produced using a targeted UHPLC-MS/MS reference method.

The issue of variability introduced into blood plasma and serum analysis by preanalytical procedures is the major obstacle to obtaining accurate and reproducible results. While the question of how to overcome this issue has been discussed in biochemical detection of analytes and omics technologies, its relevance to the field of optical spectroscopy remains mostly unexplored. In this work, we evaluated the freeze–thaw cycle (FTC)-induced alternations in blood serum optical properties by means of autofluorescence and Raman spectroscopy, including surface-enhanced Raman spectroscopy (SERS). In the case of regular Raman spectroscopy, FTC-specific spectral variability was estimated to be <1%, being significantly smaller than patient-specific variability, while the t-distributed stochastic neighbor embedding clustering of principal components yielded spectral grouping by patient ID independent of sample freezing. For SERS, FTC-specific and patient-specific spectral variabilities were 15% and >90%, respectively. Finally, parallel factor analysis of autofluorescence excitation–emission matrices revealed that patient-specific variability in the visible spectral range was 13%, whereas FTC-specific variability was 4%. We further evaluated disease-specific variability for two datasets, namely, for colorectal cancer diagnostics with autofluorescence and for chronic kidney disease diagnostics using SERS. Disease-associated variabilities were determined to be 8% and 49%, significantly exceeding the possible FTC-induced variability. Hence, the obtained results suggest that FTC blood serum samples can be used for disease diagnostics by Raman spectroscopy and SERS, as well as through autofluorescence spectroscopy, although the difference in FTC-induced and disease-induced variabilities was lowest in the latter case.

In this research, we fabricated a sensitive monoclonal antibody (mAb) 2C3 that targeted etomidate (ET) and metomidate (MT) to establish a lateral-flow immunoassay (LFIA) that incorporated fluorescent microsphere sensors, enabling both the qualitative and quantitative detection of ET and MT within 10 min. Analysis indicated that the visual colorimetric values for ET and MT in water samples were 0.3 μg kg⁻¹, respectively, with quantitative detection ranges of 0.08 to 1.31 μg kg⁻¹ and 0.08 to 2.21 μg kg⁻¹, respectively. The visual colorimetric values for ET and MT in urine samples were 0.3 and 1 μg kg⁻¹, respectively, with quantitative detection ranges of 0.12 to 3.13 μg kg⁻¹ and 0.09 to 2.98, respectively, while for ET and MT in serum samples were 3 μg kg⁻¹, with quantitative detection ranges of 0.15 to 21.2 μg kg⁻¹ and 0.08 to 13.8 μg kg⁻¹, respectively. Recovery tests and detection were conducted in water, urine and serum samples, validating the reliability of this method in clinical samples, consistent with those obtained from LC-MS/MS. Collectively, our novel LFIA provides a promising option for rapid on-site detection of ET and MT.

Latent fingerprints (LFPs) are invisible impressions that need to be developed before being used for criminal investigation; however, existing fingerprint visualization techniques face challenges, such as complex preparation and poor contrast. To advance practical fingerprint detection, green-emissive micron-sized curcumin/kaolin composites were synthesized via a facile and cost-effective one-step physical cross-linking method, which exhibited unprecedented performance in developing diversified marks, including LFPs, knuckle prints, palm prints, and footprints, with clear three-level details on various substrates. Notably, the powders successfully developed LFPs that were aged for 30 days and even up to 100 days, meeting the stringent requirements for comprehensive forensic application. Afterward, a novel method, termed Fingerprint Fluorescence Intensity Ratio (FFIR), was developed to quantify the contrast between fingerprint signals and background noise and to compare the efficacy of full-color developing agents. Compared with the existing grayscale conversion strategy, the proposed FFIR method achieved tunable multi-color fingerprint image enhancement for the first time, which helped to eliminate background fluorescence interference and improved visual perception. The feasibility of FFIR and its sensitivity in tracking image capture parameters were demonstrated by the established mathematical model. Hence, the newly synthesized modified composites and the mathematical model-validated method demonstrate profound practical significance in comprehensive fingerprint imaging.

As a marker of human metabolism, acetone is important for lipid metabolism monitoring and early detection of diabetes. In this study, we developed a handheld biosensor for acetone based on fluorescence detection by utilizing the enzymatic reaction of secondary alcohol dehydrogenase (S-ADH) with β-nicotinamide adenine dinucleotide (NADH, λ_ex = 340 nm, λ_em = 490 nm). In the reaction, NADH is oxidized when acetone is reduced to 2-propanol by S-ADH, and the acetone concentration can be measured by detecting the amount of NADH consumed in this reaction. First, we constructed a compact and light-weight fluorometric NADH detection system (209 g for the sensing system and 342 g for the PC), which worked using battery power. Then, sensor characteristics were evaluated after optimization of the working conditions. The developed system was able to quantify acetone in a range of 510 nM–1 mM within 1 minute. The developed battery-operated acetone biosensor demonstrated its ability to measure the acetone concentration in the exhaled breath condensate of 10 healthy subjects at rest (23.4 ± 15.1 μM) and after 16 h of fasting (37.7 ± 14.7 μM) and it distinguished the results with significant differences (p = 0.011). With the advantages of handheld portability, and high levels of sensitivity and selectivity, this sensor is expected to be widely used in clinical diagnosis and wearable biochemical sensors in the future.

Infrared spectro-microscopy is a powerful technique for analysing chemical maps of cells and tissues for biomedical and clinical applications, yet the strong water absorption in the mid-infrared region is a challenge to overcome, as it overlaps with the spectral fingerprints of biological components. Microfluidic chips offer ultimate control over the water layer thickness and are increasingly used in infrared spectro-microscopy. However, the actual impact of the water layer thickness on the instrument's performance is often left to the experimentalist's intuition and the peculiarities of specific instruments. Aiming to experimentally test the amount of absorption introduced by water with varying layer thicknesses, we fabricated a set of microfluidic devices with three controlled chamber thicknesses, each comprising a simple test pattern made of a well-known photoresist SU-8. We employed two infrared spectro-microscopy methods for measurements. The first method involves using a standard FTIR microscope with a benchtop infrared light source. The second method is a quantum infrared microscopy technique, where infrared imaging is achieved by detecting correlated photons in the visible range. We demonstrated that both methods enable the measurement of the absorption spectrum in the mid-IR region, even in the presence of up to a 30 μm thick water layer on top of a sample pattern. Additionally, the Q-IR technique offers practical advantages over synchrotron-based FTIR, such as reduced complexity, cost, and ease of operation.

High throughput intracellular delivery of biological macromolecules is crucial for cell engineering, gene expression, therapeutics, diagnostics, and clinical studies; however, most existing techniques are either contact-based or have throughput limitations. Herein, we report a light-activated, contactless, high throughput photoporation method for highly efficient and viable cell transfection of more than a million cells within a minute. We fabricated reduced graphene oxide (rGO) nanoflakes that was mixed with a polydimethylsiloxane (PDMS) nanocomposite thin sheet with an area of 3 cm² and a thickness of ∼600 μm. Upon infrared (980 nm) nanosecond pulse laser exposure, the rGO nanoflakes induced heat and created photothermal bubbles, leading to cell membrane deformation and biomolecular delivery. Using this platform, we achieved delivery of small to large size molecules, such as propidium iodide (PI) dye (668 Da), dextran (3000 Da), siRNA (20–24 bp), EGFP (6159 bp) and enzymes (465 kDa), in L929, N2a, and HeLa cells as well as in hard-to-transfect NiH3T3 and HuH7 cells. The best results were achieved for enzymes with ∼97% transfection efficiency and 98% cell viability in Huh7 cells. This highly efficient cargo delivery tool is simple and easy to use, and its dimensions can be varied according to the user requirements. Moreover, this safe and successful method has applicability in diagnostics and cell therapy.

MicroRNAs (miRNAs) are considered reliable biomarkers for a variety of diseases. However, their low abundance in organisms and high sequence similarity of homologous miRNAs make their accurate detection challenging. Here, we constructed a novel fluorescent biosensor for the detection of miRNA-155, a potential biomarker of neuroinflammation, based on duplex-specific nuclease (DSN) assisted amplification and DNA-templated silver nanoclusters (DNA-AgNCs) as fluorescence signal probes. DSN-assisted amplification can transform unstable miRNA into stable DNA and amplify the miRNA signal at the same time. Using DNA-AgNCs as fluorescence signal probes for biosensors can avoid complex labeling processes and reduce costs. The biosensor shows excellent selectivity, reproducibility, a wide linear range (1–600 nM) with a detection limit of 0.86 nM, and potentiality for real sample detection. This work provides a potential universal biosensing platform for miRNA detection.

Most of the existing SERS systems failed to achieve satisfactory results in early diagnosis of Alzheimer's disease owing to a lack of effective signal transduction. Herein, we developed a dual signal amplification strategy for SERS detection of amyloid-β oligomers based on proximity hybridization-triggered catalyzed hairpin assembly (CHA) and hybridization chain reaction (HCR). In the presence of the target protein and two DNA-labeled antibodies, a proximate complex formed in a homogeneous solution. Each of the AβO-DNA complexes served as a catalyst to trigger and accelerate numerous hybridization processes between MB1 and MB2. Subsequently, the single-strand fragment on the electrode surface initiated HCR, resulting in the hybridization reaction to form double-strand DNA concatemers on the substrate surface. The surface became negatively charged and allowed the absorption of silver ions on the DNA skeleton. After chemical reduction by hydroquinone, the formed silver nanoparticles could be further grown with a silver enhancement step to amplify the detectable SERS signal by absorbing rhodamine 6G as a SERS reporter on the silver nanoparticle surface. This biosensing platform had potential applications in molecular diagnostics of AD serum samples.