On-Chip Capture, Raman-Silent Polymer Labeling, and Digital Mapping Analysis of Escherichia coli O157:H7 in Beverages All-in-One.

Escherichia coli O157:H7 is one of the most susceptible foodborne pathogens, easily causing food poisoning and other health risks. It is of great significance to establish a quantitative method with higher sensitivity and less time consumption for foodborne pathogens analysis. The Raman-silent signal has a good performance for avoiding interference from the food matrix so as to achieve accurate signal differentiation. In this work, we presented a preparation-mapping all-in-one method for digital mapping analysis. We prepared a functionalized Raman-silent polymer label of Escherichia coli O157:H7, which was captured on a porous 4-mercaptophenylboric acid@Ag foam chip. To improve accuracy and widen the detection range, a digital mapping quantitative strategy was employed in data extraction and processing. By transfer mapping information into digitized statistical results, the limitation of obtaining reproducible intensity values just by randomly selected spots on the substrate can be addressed. With a wide linear range of 1.0 × 101-1.0 × 105 CFU mL-1 and a limit of detection of 4.4 CFU mL-1, this all-in-one method had good sensitivity performance. Also, this method achieved good precision and selectivity in a series of experiments and was successfully applied to the analysis of beverage samples.

Liquid-Phase Cyclic Chemiluminescence for the Identification of Cobalt Speciation.

Accurate discrimination of metal species is a significant analytical challenge. Herein, we propose a novel methodology based on liquid-phase cyclic chemiluminescence (CCL) for the identification of cobalt speciation. The CCL multistage signals (In) of the luminol-H2O2 reaction catalyzed by different cobalt species have different decay coefficients k. Thereby, we can facilely identify various cobalt species according to the distinguishable k values, including the complicated and structurally similar cobalt complexes, such as analogues of [Co(NH3)5X]n+ (X = Cl-, H2O, and NH3), Co(II) porphyrins, and bis(2,4-pentanedione) cobalt(II) derivatives. Especially, the number of substituent atoms also influences the k value greatly, which allows excellent discrimination between complexes that only have a subtle difference in the substituent group. In addition, linear discriminant analysis based on In provides a complementary solution to improve the differentiating ability. We performed density functional theory calculations to investigate the interaction mode of H2O2 over cobalt species. A close negative correlation between the adsorption energy and the k value is observed. Moreover, the calculation of energy evolutions of H2O2 decomposition into a double hydroxide radical shows that a high level of consistency exists between the activation energy barrier and the k value. The results further demonstrate that the decay coefficient of the CCL multistage signal is associated with the catalytic reactivity of the cobalt species. Our work not only broadens the application of chemiluminescence but also provides a complementary technology for speciation analysis.

Synergetic Multichiral Covalent Organic Framework for Enantioselective Recognition and Separation.

In enantiomer recognition and separation, a highly enantioselective approach with universal applicability is urgently desired but hard to realize, especially in the case of chiral molecules. To resolve the trade-off between enantioselectivity and universality, a glutathione (GSH) and methylated cyclodextrins (MCD)-functionalized covalent organic framework (GSH-MCD COF) with porosity and abundant chiral surfaces is presented that was designed and synthesized for recognition and separation of various enantiomers. As expected, the GSH-MCD COF can be used as chiral stationary phases for the separation of various enantiomers, including aromatic alcohols, aromatic acids, amides, amino acids, and organic acids, with performance and versatility even superior to some widely used commercial chiral chromatographic columns. Furthermore, the synthesized GSH-MCD COF shows high enantioselectivity for the rapid recognition and identification of enantiomers and chiral metabolites when coupling to Raman spectroscopy. Molecular simulations suggest that the COF provides a confined microenvironment for cyclodextrins and peptides that dictates the separation and recognition capability. This work provides a strategy to synthesize synergetic multichiral COF and achieve separations and recognitions of enantiomers in complex samples.

Bifunctional Mo2N Nanoparticles with Nanozyme and SERS Activity: A Versatile Platform for Sensitive Detection of Biomarkers in Serum Samples.

The combined application of nanozymes and surface-enhanced Raman scattering (SERS) provides a promising approach to obtain label-free detection. However, developing nanomaterials with both highly efficient enzyme-like activity and excellent SERS sensitivity remains a huge challenge. Herein, we proposed one-step synthesis of Mo2N nanoparticles (NPs) as a "two-in-one" substrate, which exhibits both excellent peroxidase (POD)-like activity and high SERS activity. Its mimetic POD activity can catalyze the 3,3',5,5'-tetramethylbenzidine (TMB) molecule to SERS-active oxidized TMB (ox-TMB) with high efficiency. Furthermore, combining experimental profiling with theory, the mechanism of POD-like activity and SERS enhancement of Mo2N NPs was explored in depth. Benefiting from the outstanding properties of Mo2N NPs, a versatile platform for indirect SERS detection of biomarkers was developed based on the Mo2N NPs-catalyzed product ox-TMB, which acts as the SERS signal readout. The feasibility of this platform was validated using glutathione (GSH) and target antigens alpha-fetoprotein antigen (AFP) and carcinoembryonic antigen (CEA) as representatives of small molecules with a hydroxyl radical (·OH) scavenging effect and proteins with a low Raman scattering cross-section, respectively. The limits of detection of GSH, AFP, and CEA were as low as 0.1 µmol/L, 89.1, and 74.6 pg/mL, respectively. Significantly, it also showed application in human serum samples with recoveries ranging from 96.0 to 101%. The acquired values based on this platform were compared with the standard electrochemiluminescence method, and the relative error was less than ±7.3. This work not only provides a strategy for developing highly active bifunctional nanomaterials but also manifests their widespread application for multiple biomarkers analysis.

A Portable Array Visualization Device Integrating Sample Preparation and Detection All-in-One for the On-Site Analysis of Complex Samples.

A gas membrane separation/array fluorescence visualization (GMS/AFV) device is developed by integrating hydrazine-based carbonized copolymer dots (PD-N2H4) for visual on-site analysis. The novel PD-N2H4 was synthesized using a "polymer template" approach, exhibiting strong blue fluorescence capable of visual sensing. The GMS/AFV device integrates sample preparation and detection all-in-one, consisting of a smartphone, a sample pretreatment system, and an optical system. In the detection procedure, the samples will be treated in the sample pretreatment system to create volatile gases. Therefore, any gas samples as well as solid and liquid samples that potentially produce volatile gases can be visually detected on-site by the device. H2S was utilized as a model analyte to test the practicality of the GMS/AFV device. The entire analysis can be finished in 3 min, and the limit of detection of H2S is as low as 3.4 µg/L. Surprisingly, the device is also capable of high-throughput sample detection, which can process 48 samples simultaneously in about 20 min. The device offers a quick, easy, cheap, and environmentally friendly way to analyze volatile gases, and it creates new opportunities for on-site detection of complex samples.

Recent Progress on Microfluidics Integrated with Fiber-Optic Sensors for On-Site Detection.

This review introduces a micro-integrated device of microfluidics and fiber-optic sensors for on-site detection, which can detect certain or several specific components or their amounts in different samples within a relatively short time. Fiber-optics with micron core diameters can be easily coated and functionalized, thus allowing sensors to be integrated with microfluidics to separate, enrich, and measure samples in a micro-device. Compared to traditional laboratory equipment, this integrated device exhibits natural advantages in size, speed, cost, portability, and operability, making it more suitable for on-site detection. In this review, the various optical detection methods used in this integrated device are introduced, including Raman, ultraviolet-visible, fluorescence, and surface plasmon resonance detections. It also provides a detailed overview of the on-site detection applications of this integrated device for biological analysis, food safety, and environmental monitoring. Lastly, this review addresses the prospects for the future development of microfluidics integrated with fiber-optic sensors.

Ag/Poly(N-isopropylacrylamide)-laponite Hydrogel Surface-Enhanced Raman Membrane Substrate for Rapid Separation, Concentration and Detection of Hydrophilic Compounds in Complex Sample All-in-One.

In this work, Ag nanoparticles (AgNPs) were embedded into a poly(N-isopropylacrylamide)-laponite (Ag/PNIP-LAP) hydrogel membrane for highly sensitive surface-enhancement Raman scattering (SERS) detection. In situ polymerization was initiated by UV light to encapsulate AgNPs in a PNIP-LAP hydrogel to prepare a highly active SERS membrane with a three-dimensional structure. Due to the surface plasmon resonance and high swelling/shrinkage ratio of the Ag/PNIP-LAP hydrogel SERS membrane, its network structure has a "sieving" effect, which makes it easier for hydrophilic small-molecule targets to enter the sterically confined hydrogel, and the AgNPs are close to each other to form a Raman "hot spot" through the shrinkage of the hydrogel, at the same time, the analyte is enriched in the confined space and close to the AgNPs to form a stronger SERS signal. The characterization of the SERS activity of the Ag/PNIP-LAP hydrogel showed that the prepared three-dimensional membrane had high detection sensitivity for urotropine, 2,5-dimethylpyrazine, pyrazinamide, and pyrazine; the detection limits (S/N = 3) were 17.4, 31.0, 53.1, and 1.11 µg/L, and the analytical time was 35 min. Due to the hydrophilicity of the Ag/PNIP-LAP hydrogel membrane, the small molecules can easily enter the SERS membrane, and the hydrophobic macromolecules are blocked outside the SERS membrane. The SERS method has good selectivity, stability, and reproducibility. The SERS method was applied to the detection of urotropine in dried bean curd sticks, 2,5-dimethylpyrazine in nuts and potato chips, and pyrazinamide in human plasma with recoveries of 81.8-116.8% and the relative standard deviation within 4.9-9.9%. The results matched well with that found by the corresponding chromatographic methods. The proposed method has the advantages of simple sample pretreatment, speediness, high sensitivity, and good selectivity to hydrophilic compounds and has potential application in the rapid on-site detection.

Enrichment-Sensing All-in-One Strategy Integrated in the La(OH)3-Au@AgNPs Substrate for Rapid Surface-Enhanced Raman Spectroscopy Analysis of Purine Components.

Improving the speediness of complex sample analysis has attracted much research interest in analytical science. In this work, an enrichment-sensing all-in-one strategy was presented for rapid surface-enhanced Raman spectroscopy (SERS) analysis of purine components by using the La(OH)3-Au@AgNPs nanocomposite. Two-dimensional (2D) La(OH)3 nanosheets with nanothickness and accessible active sites not only acted as efficient media for the rapid enrichment of analytes but also provided flat planes for the intensive decoration of Au@AgNPs nanoparticles to amplify the SERS signals of adsorbed analytes. The nanocomposite could realize the rapid enrichment-sensing of purine components in 1 min, including mercaptopurine, thioguanine, adenine, and purine. Subsequently, the surface adsorption behaviors were explored by density functional theory and the enhancement mechanisms were simulated by the finite-difference time-domain method. Moreover, the nanocomposite also exhibited good SERS performances with relative standard deviations (RSDs) of uniformity less than 6.5% (n = 23), RSDs of batch-to-batch stability less than 7.3% (n = 9), and long-term stability over 9 weeks with RSDs within 6.6%. Finally, the enrichment-sensing strategy was applied for the rapid SERS analysis of two projects: mercaptopurine in tablets and adenine in beers with detection limits of 6.0 and 0.76 µg/L and spiked recoveries of 90.9-100 and 84.2-101%, respectively. Benefiting from the high-performance enrichment medium and closely packed plasmonic nanoparticles, the enrichment-sensing all-in-one strategy possesses great potential for rapid on-site detection in food safety and pharmaceutical analysis.

Contractile Hairpin DNA-Mediated Dual-Mode Strategy for Simultaneous Quantification of Lactoferrin and Iron Ion by Surface-Enhanced Raman Scattering and Fluorescence Analysis.

DNA-mediated self-assembly technology with good sensitivity and affinity ability has been rapidly developed in the field of probe sensing. The efficient and accurate quantification of lactoferrin (Lac) and iron ions (Fe3+) in human serum and milk samples by the probe sensing method can provide useful clues for human health and early diagnosis of anemia. In this paper, contractile hairpin DNA-mediated dual-mode probes of Fe3O4/Ag-ZIF8/graphitic quantum dot (Fe3O4/Ag-ZIF8/GQD) NPs were prepared to realize the simultaneous quantification of Lac by surface-enhanced Raman scattering (SERS) and Fe3+ by fluorescence (FL). In the presence of targets, these dual-mode probes would be triggered by the recognition of aptamer and release GQDs to produce FL response. Meanwhile, the complementary DNA began to shrink and form a new hairpin structure on the surface of Fe3O4/Ag, which produced hot spots and generated a good SERS response. Thus, the proposed dual-mode analytical strategy possessed excellent selectivity, sensitivity, and accuracy due to the dual-mode switchable signals from "off" to "on" in SERS mode and from "on" to "off" in FL mode. Under the optimized conditions, a good linear range was obtained in the range of 0.5-100.0 µg/L for Lac and 0.01-5.0 µmol/L for Fe3+ and with detection limits of 0.14 µg/L and 3.8 nmol/L, respectively. Finally, the contractile hairpin DNA-mediated SERS-FL dual-mode probes were successfully applied in the simultaneous quantification of iron ion and Lac in human serum and milk samples.

Chemical-Chemical Redox Cycle Signal Amplification Strategy Combined with Dual Ratiometric Immunoassay for Surface-Enhanced Raman Spectroscopic Detection of Cardiac Troponin I.

Improving the sensitivity and reproducibility of surface-enhanced Raman spectroscopy (SERS) methods for the detection of bioactive molecules is crucial in biological process research and clinical diagnosis. Herein, we designed a novel SERS platform for cardiac troponin I (cTnI) detection by a chemical-chemical redox cycle signal amplification strategy combined with a dual ratiometric immunoassay. First, ascorbic acid (AA) was generated by enzyme-assisted immunoreaction with a cTnI-anchored sandwich structure. Then, oxidized 4-mercaptophenol (ox4-MP) was reacted with AA to produce 4-mercaptophenol (4-MP). Quantitative analysis of cTnI was realized by a Raman signal switch between ox4-MP and 4-MP. Specifically, AA could be regenerated by reductant (tris(2-carboxyethyl) phosphine, TCEP), which in turn produced more signal indicator 4-MP, causing significant signal amplification for cTnI analysis by SERS immunosensing. Moreover, a dual ratiometric-type SERS method was established with the intensity ratio I1077/I822 and I633/I822, which improved the reproducibility of the cTnI assay. The excellent performance of the chemical-chemical redox cycle strategy and ratio-type SERS assay endows the method with high sensitivity and reproducibility. The linear ranges of cTnI were 0.001 to 50.0 ng mL-1 with detection limits of 0.33 pg mL-1 (upon I1077/I822) and 0.31 pg mL-1 (upon I635/I822), respectively. The amount of cTnI in human serum samples yielded recoveries from 89.0 to 114%. This SERS method has remarkable analytical performance, providing an effective approach for the early diagnosis of cardiovascular diseases, and has great latent capacity in the sensitive detection of bioactive molecules.

Chiral-Controlled Cyclic Chemiluminescence Reactions for the Analysis of Enantiomer Amino Acids.

The similarity and complexity of chiral amino acids (AAs) in complex samples remain a significant challenge in their analysis. In this work, the chiral metal-organic framework (MOF)-controlled cyclic chemiluminescence (CCL) reaction is developed and utilized in the analysis of enantiomer AAs. The chiral MOF of d-Co0.75Zn0.25-MOF-74 is designed and prepared by modifying the Co0.75Zn0.25-MOF-74 with d-tartaric acid. The developed chiral bimetallic MOF can not only offer the chiral recognize sites but also act as the catalyst in the cyclic luminol-H2O2 reaction. Moreover, a distinguishable CCL signal can be obtained on enantiomer AAs via the luminol-H2O2 reaction with the control of d-Co0.75Zn0.25-MOF-74. The amplified difference of enantiomer AAs can be quantified by the decay coefficient (k-values) which are calculated from the exponential decay fitting of their obtained CCL signals. According to simulation results, the selective recognition of 19 pairs of AAs is controlled by the pore size of the MOF-74 and their hydrogen-bond interaction with d-tartaric acid on the chiral MOF. Furthermore, the k-values can also be used to estimate the change of chiral AAs in complex samples. Consequently, this chiral MOF-controlled CCL reaction is applied to differentiate enantiomer AAs involved in the quality monitoring of dairy products and auxiliary diagnosis, which provides a new approach for chiral studies and their potential applications.

Size-resolved counting of circulating tumor cells on pinched flow-based microfluidic cytometry.

Precise cell detecting and counting is meaningful in circulating tumor cells (CTCs) analysis. In this work, a simple cyclic olefin copolymer (COC) microflow cytometer device was developed for size-resolved CTCs counting. The proposed device is constructed by a counting channel and a pinched injection unit having three channels. Through injection flow rate control, microspheres/cells can be focused into the centerline of the counting channel. Polystyrene microspheres of 3, 9, 15, and 20 µm were used for the microspheres focusing characterization. After coupling to laser-induced fluorescence detection technique, the proposed device was used for polystyrene microspheres counting and sizing. A count accuracy up to 97.6% was obtained for microspheres. Moreover, the proposed microflow cytometer was applied to CTCs detecting and counting. To mimic blood sample containing CTCs and CTCs mixture with different subtypes, an MDA-MB-231 (human breast cell line) spiked red blood cells sample and a mixture of MDA-MB-231 and MCF-7 (human breast cell line) sample were prepared, respectively, and then analyzed by the developed pinched flow-based microfluidic cytometry. The simple fabricated and easy operating COC microflow cytometer exhibits the potential in the point-of-care clinical application.

Recent progress of microfluidic sample preparation techniques.

Sample preparation turns out to be one of the important procedures in complex sample analysis by affecting the accuracy, selectivity, and sensitivity of analytical results. However, the majority of the conventional sample preparation techniques still suffer from time-consuming and labor-intensive operations. These shortcomings can be addressed by reforming the sample preparation process in a microfluidic manner. Inheriting the advantages of rapid, high efficiency, low consumption, and easy integration, microfluidic sample preparation techniques receive increasing attention, including microfluidic phases separation, microfluidic field-assisted extraction, microfluidic membrane separation, and microfluidic chemical conversion. This review overviews the progress of microfluidic sample preparation techniques in the last 3 years based on more than 100 references, we highlight the implementation of typical sample preparation methods in the formats of microfluidics. Furthermore, the challenges and outlooks of the application of microfluidic sample preparation techniques are discussed.

One-pot synthesis of pillar[5]quinone-amine polymer coated silica as stationary phase for high-performance liquid chromatography.

To expand the application of pillararene in chromatographic separation, we designed and fabricated a pillar[5]quinone-amine polymer coated silica through quinone-amine reaction by facile one-pot synthesis method, which was applied as a stationary phase for high-performance liquid chromatography. Separation of hydrophobic compounds, hydrophilic compounds, halogenated aromatic compounds, and 11 aromatic positional isomers was achieved successfully in this stationary phase. Reverse-phase separation mode and hydrophilic-interaction separation mode were proved to exist, indicating the potential application of the mix-mode stationary phase. Studies of chromatographic retention behavior and molecular simulation showed that multiple interactions might play an important role in the separation process, including hydrophobic interaction, hydrogen-bonding interaction, aromatic π-π interaction, electron donor-acceptor interaction, and host-guest interaction. Column repeatability and stability were tested, which showed relative standard deviations of retention time less than 0.2% for continuous 11 injections, and the durability relative standard deviations of retention time were less than 0.91% after 90 days. This novel design strategy would broaden the application of pillararene-based covalent organic polymer in chromatography and separation science.

Primary amide-functionalized cyclotricatechylene covalent organic frameworks membrane for efficient enrichment of melamine and its derivatives in migration solution of food contact materials.

A highly chemically stable primary amide-functionalized cyclotricatechylene covalent organic framework was synthesized by an irreversible reaction and a post-synthetic modification. It possessed a rod-like morphology and exhibited strong solvent stability owing to the polyether bonds. The material showed good adsorption performance for melamine and its derivatives and adsorption mechanism was investigated by molecular simulations. The adsorbent was coated on the nylon-66 membrane to prepare the enrichment membrane. Under optimized conditions, an in-syringe membrane-based extraction method, combined with ultra-high performance liquid chromatography-tandem mass spectrometry was developed for the analysis of melamine and six melamine derivatives in the migration solution. A good linearity was obtained with correlation coefficients ranging from 0.9924 to 0.9995. The limits of detection were 1-200 ng/L and the limits of quantification were 3-500 ng/L. This method was successfully applied to the migration solution of sushi bamboo rolling mats with spiked recoveries of 73.2%-115% and relative standard deviations of 0.9%-9.9%. This work shows a practical and perspective approach for the efficient enrichment of food contact material hazards.

Chemiluminescence method based on the KIO4 -K2 CO3 -Mn2+ reaction for rapid and sensitive determination of forchlorfenuron in dried fruit.

Forchlorfenuron is a low-toxic phenylurea plant growth regulator. Excessive intake of forchlorfenuron can lead to metabolic disorders of the matrix and be harmful to human health. The chemiluminescence intensity of the KIO4 -K2 CO3 -Mn2+ reaction decreased in the presence of forchlorfenuron. Based on this result, a rapid and sensitive chemiluminescence method was established to determine forchlorfenuron by combining it with a batch injection static device. The injection speed, injection volume and reagent concentration of the forchlorfenuron-KIO4 -K2 CO3 -Mn2+ chemiluminescence reaction were optimized. Under these optimized conditions, the linear range of the method was 1.0-200.0 µg/L, and the limit of detection was 0.29 µg/L (S/N = 3). The chemiluminescence method for the determination of forchlorfenuron could be completed in 10 s. The method was applied to detect the residual forchlorfenuron in dried fruit samples, and the results are consistent with high-performance liquid chromatography-mass spectrometry. This method has the advantages of high sensitivity, rapid response, less reagent consumption, and convenient operation. It will provide a new perspective for chemiluminescence for the rapid and sensitive determination of forchlorfenuron in various complex samples.

Determination of acetic acid in enzymes based on the cataluminescence activity of graphene oxide-supported carbon nanotubes coated with NiMn layered double hydroxides.

A cataluminescence (CTL) method has been developed for the rapid determination of acetic acid in enzyme products. The NiMn LDH/CNT/GO was synthesized based on the nanohybridization of NiMn layered double hydroxide (NiMn LDH), carbon nanotubes (CNTs), and graphene oxide (GO). The composite has excellent CTL activity against acetic acid. It could be ascribed to the larger specific surface area and more exposure to active sites. NiMn LDH/CNT/GO is used as a catalyst in the CTL method based on its special structure and advantages. There is a linear relationship between CTL response and the acetic acid concentration in the range 0.31-12.00 mg·L-1 with the detection limit of 0.10 mg·L-1. The developed method is rapid and takes only about 13 s. The method is applied to the determination of acetic acid in enzyme samples with little sample preparation. The result of the CTL method shows good agreement with that of the gas chromatography method. The proposed CTL method possesses promising potential in the quality monitoring of enzymes.

All-in-One Preparation Strategy Integrated in a Miniaturized Device for Fast Analyses of Biomarkers in Biofluids by Surface Enhanced Raman Scattering.

Complex and tedious sample preparation processes have greatly limited rapid analyses of biological samples. In this work, an all-in-one sample preparation strategy based on a miniaturized gas membrane separation/oven ring enrichment (GMS/ORE) device was developed for efficient surface enhanced Raman scattering (SERS) analyses of trace biomarkers in biofluid samples. This strategy integrating gasification separation, liquid trapping, derivatization SERS activation, and coffee-ring enrichment could highly promote the efficiency of sample preparation. Meanwhile, the edges of membranes modified by the hydrophobic-infusing slippery liquid-induced uniform "coffee-ring" effect could significantly improve the sensitivity and stability for SERS quantification. By adapting proper derivatization approaches to the miniaturized GMS/ORE pretreatment, the matrix effects in samples could be prominently eliminated, and clear SERS responses could be obtained for the selective analyses of target biomarkers. The miniaturized GMS/ORE device was practically applied for SERS analyses of trace biomarkers in biofluids, including hydrogen sulfide in saliva samples, creatinine in serum samples, and sarcosine, creatinine, and dimethyl disulfide in urine samples. Accurate quantification of all biomarkers was achieved with recoveries of 89.5%-120.0%, and the contents found by GMS/ORE-SERS matched well with those found by corresponding chromatographic methods with relative errors from -8.6% to 9.3%. The miniaturized GMS/ORE device with multiple parallel processing units could simultaneously treat eight samples in one run with a total analysis time of 40 min. Such an efficient all-in-one strategy integrated on a miniaturized device possesses great potential for fast on-site/point-of-care detection in analytical science and clinical medicine.

Microfluidic Magnetic Spatial Confinement Strategy for the Enrichment and Ultrasensitive Detection of MCF-7 and Escherichia coli O157:H7.

A microfluidic magnetic spatial confinement strategy was developed and employed to realize an ultrasensitive cell immunoassay. The straight confined channels in poly(dimethylsiloxane)-glass hybrid microchips were used as the enrichment and detection chambers for the proposed microfluidic magnetic cell immunoassays (µMCI). To accomplish the µMCI, prepared magnetic cell immunocomplexes were introduced into microchannels and preconcentrated in the detection zone under a permanent magnet. The magnetic cell immunocomplexes were constructed from aptamer-/antibody-coated magnetic beads and antibody-linked horseradish peroxidase-labeled target cells to guarantee the specificity and enhance the detection signal generated from the enzyme reaction. The sensitivity enhancement of µMCI was confirmed in a one-dimensional space confined microchamber, especially in the analysis of cells having more enzyme conjugating sites on their surface. This spatial confinement strategy based µMCI was then applied for model cell detection in the microchannel, the limits of detection (LODs) were 2 cells/mL for MCF-7 and 34 colony-forming unit/mL for Escherichia coli O157:H7 (E. coli O157:H7), which corresponded to up to 1202-fold LOD sensitivity improvement compared to the results of the similar immunoassays in microwell plates. The satisfactory selectivity and reproducibility of the strategy were also obtained. Moreover, it enabled rare MCF-7 detection in whole blood and E. coli O157:H7 detection in milk after time-shortened incubation. Constructing an appropriate confined space, this strategy can be extended to detect various cells with higher sensitivity, which provides a valuable approach for rare cell detection in practical applications.

ATP-Responsive Strand Displacement Coupling with DNA Origami/AuNPs Strategy for the Determination of Microcystin-LR Using Surface-Enhanced Raman Spectroscopy.

The DNA origami-mediated self-assembly strategy has emerged as a powerful tool in surface-enhanced Raman spectroscopy (SERS). However, these self-assembly approaches typically do not possess high detection specificity. Herein, a novel strategy based on adenosine triphosphate (ATP)-responsive strand displacement (ARSD) coupling with DNA origami/AuNPs for SERS analysis of microcystin-LR (MC-LR) is presented. In the presence of MC-LR and ATP molecules, nucleic acid sensing structures fabricated with anti-MC-LR aptamer (T1) and ATP aptamer (T2) were triggered to release the remaining ATP. In addition, DNA origami-assisted assembly results in the formation of homogeneous plasmonic nanostructures for Raman enhancement via strong plasmonic coupling. After the binding in the gaps of functionalized DNA origami/AuNPs, the Raman shift of the ATP molecules becomes detectable, leading to increased SERS intensity in 734 cm-1. A linear response to MC-LR was obtained in the concentration range of 1.56-50 µg·L-1, and the limit of detection (LOD) was 0.29 µg·L-1. Combined with the solid-phase extraction sample pretreatment for extraction and 10-fold concentration, this proposed method was successfully used to detect MC-LR type in real lake-water samples with good recoveries of 98.4-116% and relative standard deviations of 1.9-6.7%. Furthermore, for the detection of MC-LR in contaminated lake-water samples, the results of the developed method and ultrahigh-performance liquid chromatography-tandem mass spectrometry were found to be in agreement with relative errors between -12 and 2.4%. The proposed strategy provides a sensitive recognition and signal amplification platform for trace MC-LR analysis as well as innovative nucleic acid sensing structures for toxin analysis more generally.