Analyst最新文献

When exposed to an alternating current (AC) electric field, a polarized microparticle is moved by the interaction between the voltage-induced dipoles and the AC electric field under dielectrophoresis (DEP). The DEP force is widely used for manipulation of microparticles in diverse practical applications such as 3D manipulation, sorting, transfer, and separation of various particles such as living cells. In this study, we propose the integration of surface-enhanced Raman spectroscopy (SERS), an extremely sensitive and versatile technique based on the Raman scattering of molecules supported by nanostructured materials, with DEP using a microfluidic device. The microfluidic device combines microelectrodes with gold nanohole arrays to characterize the electrophysiological and biochemical properties of biological cells. The movement of particles, which varies depending on the electrical properties such as conductivity and permittivity of particles, can be manipulated by the cross-frequency change. For proof of concept, Raman spectroscopy using the DEP–SERS integration was performed for polystyrene beads and biological cells and resulted in an improved signal-to-noise ratio by determining the direction of the DEP force applied to the cells with respect to the applied AC power and collecting them on the nanohole arrays. The result illustrates the potential of the concept for simultaneously examining the electrical and biochemical properties of diverse chemical and biological microparticles in the microfluidic environment.

In the face of worsening water quality and escalating water environmental emergencies, this study developed a paper-based microfluidic disk for rapid, on-site determination of ammonia nitrogen, nitrates, nitrites, and phosphates in water. The method utilizes centrifugal microfluidics and paper-based technology, thus simplifying the operation while eliminating the need for on-site reagent preparation. Experimental results demonstrate that the disk requires only 80 microliters of a water sample and 2 minutes to complete the quantitative analysis of the four nutrients, with a coefficient of variation below 1.72% and spike recoveries ranging from 92% to 113%. The development of the disk provides an effective and rapid, on-site testing tool for water quality analysis.

Fluorescence labeling via fluorescent proteins (FPs) or immunofluorescence has been routinely applied for microscopic imaging of specific proteins. However, due to these over-weight and oversized labels (e.g. GFP, 238 aa, 27 kDa, ∼4 nm in size), the potential physiological malfunctions of the target proteins are largely underestimated in living cells. Herein, for living cells, we report a small and minimally-invasive Raman reporter (about 2 aa and <1 kDa), which can be site-specifically introduced into proteins by genetic codon expansion. After a single unnatural amino acid (UAA) is precisely incorporated into the target protein, the strained alkyne can rapidly undergo copper-free Diels–Alder cycloaddition reactions with the tetrazine-functionalized Raman reporter, which features a fine vibrational spectrum in contrast to fluorescence. In our experimental results, the UAA-based Raman tag was successfully incorporated into vimentin, histone 3.3 and huntingtin (Htt74Q) proteins in living HeLa cells and further utilized for stimulated Raman imaging. The site-specific bioorthogonal fusion of small Raman tags with intracellular proteins will pave the way for minimally-invasive protein labeling and multi-color imaging in living cells.

Non-specific amplification (NSA, amplification in the absence of a target analyte) in bioanalytical rolling circle amplification (RCA) assays, especially those involving pre-synthesized circular DNA (cDNA), affects its analytical sensitivity. Despite extensive development of RCA-based bioanalytical methods, the NSA in RCA remains uncharacterized in terms of its magnitude or origin. NSA may originate from inefficient ligation or succeeding cDNA purification steps. This study comprehensively quantifies NSA across several ligation and digestion techniques for the first time since the innovation of RCA. To quantify the NSA in RCA, cDNAs were prepared using self-annealing, splint-padlock, or cohesive end ligations. The cDNAs were then subjected to nine different exonuclease digestion steps and quantified for NSA under linear as well as hyperbranched RCA conditions. We investigated buffer compositions, divalent ion concentrations, single or dual enzyme digestion, cohesive end lengths, and splint lengths. The optimized conditions successfully mitigated absolute NSA by 30–100-fold and relative NSA (normalized against primer-assisted RCA) to ∼5%. Besides understanding the mechanistic origin of NSA, novel aspects of enzyme–substrate selectivity, buffer composition, and the role of divalent ions were discovered. With increasing bioanalytical RCA applications, this study will help standardize NSA-free assays.

Group A streptococcus (GAS) is a pathogen typically transmitted through respiratory droplets and skin contact, causing an estimated 700 million mild non-invasive infections worldwide each year. There are approximately 650 000 infections that progress to severe invasive infections, even resulting in death. Therefore, the ability to detect GAS rapidly, accurately and in real time is important. Herein, we developed a nanozyme linked multi-array gas driven sensor (NLMAGS) to point-of-care testing of GAS within 2 h. The NLMAGS demonstrated excellent performance as it combined the advantages of nanozyme techniques, immunoassay techniques, and 3D printing techniques. Platinum- and palladium-rich nanozyme particles (Au@Pt@PdNPs) were synthesized and used to label monocloning antibodies as detection probes. Magnetic beads were labeled with monocloning antibodies as capture probes to establish a double-antibody sandwich immunoassay for the detection of GAS. The sandwich immune complex can catalyze the H₂O₂ substrate and produce O₂. GAS quantification can be achieved by measuring the distance that the O₂ pushes the ink drops forward in the sensor. Under optimized conditions, the NLMAGS quantitatively detected 24 spiked samples with a limit of detection (LOD) of 62 CFU mL⁻¹, which was 5 times lower than that of ELISA (334 CFU mL⁻¹). A strong correlation with the conventional ELISA was found (r = 0.99, P < 0.001). In comparison, the traditional lateral flow immunoassay based on Au@Pt@PdNPs-mAb2 (Au@Pt@PdNPs-LFIA) had a LOD of 10⁴ CFU mL⁻¹, which was significantly higher than that of NLMAGS. The NLMAGS demonstrated excellent sensitivity to GAS. The intra- and inter-assay precisions of the sensor were below 15%. Overall, the established NLMAGS has promising potential as a rapid and quantitative method for detecting GAS and can also be used to detect various pathogens.

Spatially offset Raman spectroscopy (SORS) is a transformative method for probing subsurface chemical compositions in turbid media. This systematic study of Monte Carlo simulations provides closed-form characterizations of key SORS parameters, such as the distribution of spatial origins of collected Raman photons and optimal SORS geometry to selectively interrogate a subsurface region of interest. These results are unified across an extensive range of material properties by multiplying spatial dimensions by the medium's effective attenuation coefficient, which can be calculated when the absorption and reduced scattering coefficients are known from the literature or experimentation. This method of spatial nondimensionalization is validated via goodness-of-fit analysis on the aggregate models and by training a subsurface sample localization model on a heterogeneous population of materials. The findings reported here advance the understanding of SORS phenomena while providing a quantitative and widely applicable foundation for designing and interpreting SORS experiments, facilitating its application in disciplines such as biomedical, materials science, and cultural heritage fields.

A double-channel methane (CH₄) sensor was developed using a dual-pass multipass cell (DP-MPC) and a novel method that combines averaging dual-channel concentration signals with optimized detector gain configuration. This DP-MPC features two input/output coupling holes, resulting in absorption path lengths of approximately 95.8 m and 35.8 m, respectively. By optimizing the photodetector gain configuration and averaging the dual-channel concentration signals, the detection performance of the sensor was further enhanced. Allan deviation analysis indicated that after optimizing the detector gain, the measurement precision after dual-channel averaging reaches 21 ppb with an integration time of 1 s at a concentration of 2 ppm CH₄, which is approximately 1.4 times higher than the measurement precision of the long-path channel (31 ppb) and short-path channel (30 ppb). The time required to achieve a measurement precision of 21 ppb is 2.4 s for the long-path channel and 2.1 s for the short-path channel. The response speed of the dual-channel averaging is approximately 2 times that of any single channel. Meanwhile, the sensor demonstrated its stability and reliability through continuous outdoor atmospheric CH₄ measurements.

Scanning Electrochemical Microscopy (SECM) has been used as a non-invasive electrochemical technique for studying cellular processes. SECM enables the quantification of cellular metabolites in real-time providing a deeper understanding of cellular responses to external stimuli. SECM imaging of living cells requires maintaining an ideal physiological environment to ensure reliable data collection on cellular reactivity. The cellular response can be directly influenced by physicochemical parameters including cell media composition, temperature and light exposure. This research demonstrates the effect of media composition on the electrochemical current signal of adenocarcinoma cervical cancer (HeLa) cells during SECM measurements using ferrocenemethanol as a redox mediator. Investigated media that are commonly used as electrolyte, are phosphate buffered saline (PBS), and Dulbecco's modified Eagle's medium (DMEM) in the absence and presence of fetal bovine serum (FBS). In addition, this research demonstrates that fluctuating light illumination impacts the stability of the cellular electrochemical current response. Our findings reveal that media composition and illumination are important parameters that must be carefully considered and monitored during SECM live cell imaging.