Integrating Sample Pretreatment Research Articles

MOF derivatization of multifunctional nanozyme: Peroxidase-like catalytic activity combined with magnetic solid phase extraction for colorimetric detection of Hg2+

Nanozyme-based colorimetric sensing has drawn immense attention due to the rapid development of nanozyme in recent years. However, the selectivity of nanozyme-based colorimetric sensing greatly limits its subsequent practical application. It is well known that sample pretreatment can not only improve selectivity by eliminating the sample matrix interference, but also improve sensitivity by enriching trace targets. Based on the easy facile surface modification properties of nanozyme, we rationally designed nanozyme combined with sample pretreatment for colorimetric biosensing, through separation and enrichment, thereby improving the selectivity and sensitivity of the nanozyme colorimetric biosensing. As a proof of concept, the detection of Hg2+ by nanozyme-based colorimetric sensing was used as an example. Magnetic peroxidase-like nanozyme Fe3S4 was designed and synthesized. The selectivity is improved by the specific adsorption of S-Hg bond and the interference elimination after magnetic separation. In addition, the sensitivity is improved by magnetic solid-phase extraction enrichment. Our established colorimetric sensing based on Fe3S4 nanozyme integrated sample pretreatment with an enrichment factor of 100 and the limit of detection (LOD) is 26 nM. In addition, this strategy was successfully applied to detect Hg2+ in environmental water samples. Overall, the strategy showed good selectivity and sensitivity, providing a new practical method for the application of nanozyme-based biosensing in sample pretreatment.

Rational design of nanozyme with integrated sample pretreatment for colorimetric biosensing

Nanozymes have been widely used in the field of biosensing owing to their high stability, low cost, adjustable catalytic activity, and convenient modification. However, achieving high selectivity and sensitivity simultaneously in nanozyme-based colorimetric sensing remains a major challenge. Nanozymes are nanomaterials with enzyme-simulating activity that are often used as solid-phase adsorbents for sample pretreatment. Our design strategy integrated sample pretreatment function into the nanozyme through separation and enrichment, thereby improving the selectivity and sensitivity of nanozyme-based colorimetric biosensing. As a proof-of-concept, glucose was used as the model analyte in this study. A phenylboric acid-modified magnetic nanozyme (Cu/Fe3O4@BA) was rationally designed and synthesized. Selectivity was enhanced by boronate-affinity specific adsorption and the elimination of interference after magnetic separation. In addition, magnetic solid-phase extraction enrichment was used to improve the sensitivity. A recovery rate of more than 80% was reached when the enrichment factor was 50. The synthesized magnetic Cu/Fe3O4@BA was recyclable at least five times. The proposed method exhibited excellent selectivity and sensitivity, simple operation, and recyclability, providing a novel and practical strategy for designing multifunctional nanozymes for biosensing.

Dual-Track Multifunctional Bimetallic Metal-Organic Frameworks for Antibiotic Enrichment and Detection.

The improper use and overuse of antibiotics have led to significant burdens and detrimental effects on the environment, food supply, and human health. Herein, a magnetic solid-phase extraction program and an optical immunosensor based on bimetallic Ce/Zr-UiO 66 for the detection of antibiotics are developed. A magnetic Fe3O4@SiO2@Ce/Zr-UiO 66 metal-organic framework (MOF) is prepared to extract and enrich chloramphenicol from fish, wastewater, and urine samples, and a horseradish peroxidase (HRP)-Ce/Zr-UiO 66@bovine serum protein-chloramphenicol probe is used for the sensitive detection of chloramphenicol based on the dual-effect catalysis of Ce and HRP. In this manner, the application of Ce/Zr-UiO 66 in integrating sample pretreatment and antibiotic detection is systematically investigated and the associated mechanisms are explored. It is concluded that Ce/Zr-UiO 66 is a versatile dual-track material exhibiting high enrichment efficiency (6.37mg g-1) and high sensitivity (limit of detection of 51.3 pg mL-1) for chloramphenicol detection and serving as a multifunctional MOF for safeguarding public health and hygiene.

High-throughput, highly sensitive and rapid SERS detection of Escherichia coli O157:H7 using aptamer-modified Au@macroporous silica magnetic photonic microsphere array.

The aim of this research was to develop a simple, rapid, sensitive, high-throughput detection method for foodborne Escherichia coli (E. coli) O157:H7 based on the aptamer-modified gold nanoparticles@macroporous magnetic silica photonic microsphere (Au@MMSPM). Such Au@MMSPM array system for E. coli O157:H7 not only integrated sample pretreatment with rapid detection, but also showed highly enhanced effect to develop a highly sensitive SERS assay. The established SERS assay platform gave a wide linear detection range (10-106 CFU/mL) and low limit of detection (2.20CFU/mL) for E. coli O157:H7. The whole analysis time including sample pretreatment and detection was 110min. This SERS-based assay platform provided a new high-throughput, highly sensitive and fast detection technology for monitoring E. coli O157:H7 in real samples from the fields of food industry, medicine and environment.

Quantitative Time-Resolved Visualization of Catalytic Degradation Reactions of Environmental Pollutants by Integrating Single-Drop Microextraction and Fluorescence Sensing.

Current methods for evaluating catalytic degradation reactions of environmental pollutants primarily rely on chromatography that often suffers from intermittent analysis, a long turnaround period, and complex sample pretreatment. Herein, we propose a quantitative time-resolved visualization method to evaluate the progress of catalytic degradation reactions by integrating sample pretreatment [single-drop microextraction, (SDME)], fluorescence sensing, and a smartphone detection platform. The dechlorination reaction of chlorobenzene derivatives was first investigated to validate the feasibility of this approach, in which SDME plays a critical role in direct sample pretreatment, and inorganic CsPbBr3 perovskite encapsulated in a metal-organic framework (MOF-5) was utilized as the fluorescent chromogenic agent (FLCA) in SDME to realize fast in situ colorimetric detection via the color switching from green (CsPbBr3) to blue (chlorine lead bromide, inorganic CsPbCl3 perovskite). The smartphone, which can calculate the B/G value of FLCA, serves as a data output window for quantitative time-resolved visualization. Further, a [Eu(PMA)]n (PMA= pyromellitic acid) fluorescent probe was constructed to use as an FLCA for the in situ evaluation of cinnamaldehyde and p-nitrophenol catalytic reduction. This approach not only minimizes the utilization of organic solvents and achieves quantitively efficient time-resolved visualization but also provides a feasible method for in situ monitoring of the progress of catalytic degradation reactions.

Fast removal of phenolic compounds from water using hierarchical porous carbon nanofibers membrane

Water pollution is becoming increasingly serious and seriously endangering human health, especially the direct emissions of phenolic compounds. An integrated sample pre-treatment and derivatization method based on a biopolymers/TEOS-based carbon nanofibers microextraction that allows rapid extraction (5 min), followed by separation and highly sensitive detection of phenolic compounds by gas chromatography‒mass spectrometry (GC-MS) analysis, is described. The developed methodology, coupled with GC-MS, allowed low detection limits (0.03–0.32 ng mL‒1), good linearities (0.5–200 ng mL‒1) and recoveries (73.58–85.76%) to be achieved in a few steps and short time. Based on the high adsorption properties of materials, the on-line removal device of phenolic compounds in water was designed. The elimination rate of phenolic compounds in water was higher than 73.58% at 1 mL min‒1 and three cycles. The elimination strategy for the phenolic compounds is very versatile and is easy to apply.

A sample-to-answer DNA detection microfluidic system integrating sample pretreatment and smartphone-readable gradient plasmonic photothermal continuous-flow PCR.

As the gold standard for nucleic acid detection, full-process polymerase chain reaction (PCR) analysis often falls into the dilemma of complex workflow, time-consuming, and high equipment costs. Therefore, we designed and optimized a DNA quantification microfluidic system by strategically integrating sample pretreatment and a smartphone-readable gradient plasmonic photothermal (GPPT) continuous-flow PCR (CF-PCR). Through preloading and sequential injection of immiscible extraction reagents, combined with magnetic bead (MB) manipulation, the microfluidic chip successfully purified and concentrated 100 μL of HBV-DNA spiked plasma into a 20-μL purified sample within 14 minutes. With a digital PCR platform, the optimized experiments showed that the DNA extraction efficiency can reach 69% at an immiscible reagent configuration ratio of 10 : 10 : 1 : 12 : 2 (sample : lysis/binding buffer : MB : silicone oil : eluent) and a flow rate of 25 μL min-1. For the first time, we used gold nanorod (AuNR)-doped PDMS to prepare a CF-PCR submodule for the amplification of a 40 μL PCR mixture. Due to the plasmonic photothermal effect of AuNRs and the gradient intensity of an expanded laser spot, the PCR thermal gradient was formed on a coin-sized area. The compact annular thermal-microfluidic layout, optimized DNA dye concentration, and chip transmittance synergistically enable a rarely reported smartphone-based fluorescence CF-PCR, greatly simplifying thermal control and detection setup. Prototype construction and validation experiments show that the microsystem can complete the sample-to-answer quantification of HBV-DNA with a dynamic linear range from 1.2 × 101 to 1.2 × 106 copies per μL in ∼37 minutes. This novel microfluidic solution effectively bridges the technical gap between the CF-PCR, sample pretreatment and result characterization, making the workflow standardized and rapid and requiring <15% of the commercial instrument cost. The simplicity, rapidity and low cost of this work make it promising for applications in decentralized laboratories and low-resource settings.

One-step integrated sample pretreatment technique by gas-liquid microextraction (GLME) to determine multi-class pesticide residues in plant-derived foods

Gas-liquid microextraction technique (GLME) has been integrated with dispersive solid phase extraction to establish a one-step sample pretreatment approach for rapid analysis of multi-class pesticides in different plant-derived foods. A 50 μL of organic solvent plus 40 mg of PSA were required throughout the 5-minute pretreatment procedure. Good trueness (recoveries of 67.2 – 105.4%) and precision (RSD ≤ 18.9%) were demonstrated by the one-step GLME method, with MLOQs ranged from 0.001 to 0.011 mg kg−1. As high as 93.6% pesticides experienced low matrix effect through this method, and the overall matrix effects (ME%) were generally better or comparable to QuEChERS. This method successfully quantified 2-phenylphenol, quintozene, bifenthrin and permethrin in the range of 0.001 – 0.008 mg kg−1 in real food samples. The multiresidue analysis feature of GLME has been validated, which displays further potential for on-site determination of organic pollutants in order to safeguard food safety and human health.

Flexible fabrication of a paper-fluidic SERS sensor coated with a monolayer of core–shell nanospheres for reliable quantitative SERS measurements

In recent years, paper-based Surface-enhanced Raman spectroscopy (SERS) substrates have received extensive attention in the field of rapid analysis. However, obtaining quantitative SERS results is still challenging because of the inferior uniformity originating from the irregular morphology of the filter paper. In this work, a novel paper-fluidic SERS sensor was developed and its in-depth applications in the real-word quantitative analysis of contaminants in complex matrices were demonstrated. In particular, the Au@Ag core-shell nanospheres were labeled with an internal standard molecule to successfully normalize the fluctuation of the SERS signal caused by the microstructure of the filter paper, which could significantly improve the detection accuracy and accomplish the SERS quantitative analysis. In addition, a facile and robust strategy for the fabrication of a paper-based SERS sensor, which uses a dropper and mask to transfer the nanoparticle monolayers, was developed. This convenient and flexible approach effectively achieved a precise patterned assembly of nanoparticles on the filter paper. Furthermore, the paper-fluidic SERS sensor was fabricated by cutting and packaging for two detection modes, i.e., lateral-flow and vertical-flow, which generates the functionalization of the paper-based SERS substrate. Both detection modes integrated sample pretreatment and sample enrichment with SERS detection were applied to accurately detect the pesticide thiram in a complex sample of orange juice with pulp. In summary, this paper-fluidic SERS sensor with a simple preparation process and integrated functions is an ideal candidate for real sample analysis without pretreatment.

Lab-on-a-disc platform for screening of genetically modified E. coli cells via cell-free electrochemical detection of p-Coumaric acid

We present a robust easy to use lab-on-a-disc (LoD) device with integrated sample pre-treatment and electrochemical detection system for cell-free detection of a secondary metabolite, p-Coumaric acid (pHCA), produced by genetically modified E. coli. In the LoD device, which incorporates eight filtration and electrochemical detection units, the sample filtration was performed by rotating the disc using a programmable closed-loop stepper motor. The electrodes, patterned on plastic substrate, were connected through a printed circuit board to the slip ring using a robust magnetic clamping system that enables easy assembly and robust electrical connections. pHCA was quantified in a linear range from 0.125 up to 2mM using square wave voltammetry. The platform was successfully used for the quantification of pHCA produced by two genetically modified E. coli strains after 24h of cell culture. The data obtained from the electrochemical measurements showed good correlation with high performance liquid chromatographic analysis. The developed LoD system offers fast and easy detection of pHCA, enabling screening of genetically modified organisms based on the quantity of produced secondary metabolites.

An integrated sample pretreatment platform for quantitative N-glycoproteome analysis with combination of on-line glycopeptide enrichment, deglycosylation and dimethyl labeling

Relative quantification of N-glycoproteomes shows great promise for the discovery of candidate biomarkers and therapeutic targets. The traditional protocol for quantitative analysis of glycoproteomes is usually off-line performed, and suffers from long sample preparation time, and the risk of sample loss or contamination due to manual manipulation. In this study, a novel integrated sample preparation platform for quantitative N-glycoproteome analysis was established, with combination of online N-glycopeptide capture by a HILIC column, sample buffer exchange by a N2-assisted HILIC–RPLC interface, deglycosylation by a hydrophilic PNGase F immobilized enzymatic reactor (hIMER) and solid dimethyl labeling on a C18 precolumn. To evaluate the performance of such a platform, two equal aliquots of immunoglobulin G (IgG) digests were sequentially pretreated, followed by MALDI-TOF MS analysis. The signal intensity ratio of heavy/light (H/L) labeled deglycosylated peptides with the equal aliquots was 1.00 (RSD=6.2%, n=3), much better than those obtained by the offline protocol, with H/L ratio as 0.76 (RSD=11.6%, n=3). Additionally, the total on-line sample preparation time was greatly shortened to 160min, much faster than that of offline approach (24h). Furthermore, such an integrated pretreatment platform was successfully applied to analyze the two kinds of hepatocarcinoma ascites syngeneic cell lines with high (Hca-F) and low (Hca-P) lymph node metastasis rates. For H/L labeled Hca-P lysates with the equal aliquots, 99.6% of log2 ratios (H/L) of quantified glycopeptides ranged from −1 to 1, demonstrating high accuracy of the developed sample preparation strategy. By triplicated analysis of glycopeptides and non-glycopeptides of Hca-F and Hca-P lysates, 43 up-regulated and 30 down-regulated (Hca-F/P) N-glycosylation sites, and 11 significantly changed N-glycoproteins were successfully quantified, and most of them were related to tumorigenesis and tumor metastasis. All these results demonstrate the developed integrated N-glycoprotein pretreatment platform is of great power for the accurate, precise and high-throughput analysis of N-glycoproteomes.

Integrated Sample Pretreatment System for N-Linked Glycosylation Site Profiling with Combination of Hydrophilic Interaction Chromatography and PNGase F Immobilized Enzymatic Reactor via a Strong Cation Exchange Precolumn

An integrated sample pretreatment system, composed of a click maltose hydrophilic interaction chromatography (HILIC) column, a strong cation exchange (SCX) precolumn, and a PNGase F immobilized enzymatic reactor (IMER), was established for the simultaneous glycopeptide enrichment, sample buffer exchange, and online deglycosylation, by which the sample pretreatment for glycoproteome could be performed online automatically, beneficial to improve the efficiency and sensitivity of the N-linked glycosylation site identification. With such a system, the deglycosylated glycopeptide from the digests of avidin with the coexistence of 50 times (mass ratio) BSA could be selectively detected, and the detection limit as low as 5 fmol was achieved. Moreover, the sample pretreatment time was significantly shortened to ~1 h. Such a system was further successfully applied for analyzing the digest of the soluble fraction extracted from rat brain. A total of 120 unique glycoprotein groups and 196 N-linked glycosylation sites were identified by nanoreversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoRPLC-ESI-MS/MS), with the injected digests amount as 6 μg. All these results demonstrate that the integrated system is of great promise for N-linked glycosylation site profiling and could be further online coupled with nanoHPLC-ESI-MS/MS to achieve high-throughput glycoproteome analysis.

Microfluidic immunoassays as rapid saliva-based clinical diagnostics

At present, point-of-care (POC) diagnostics typically provide a binary indication of health status (e.g., home pregnancy test strip). Before anticipatory use of diagnostics for assessment of complex diseases becomes widespread, development of sophisticated bioassays capable of quantitatively measuring disease biomarkers is necessary. Successful translation of new bioassays into clinical settings demands the ability to monitor both the onset and progression of disease. Here we report on a clinical POC diagnostic that enables rapid quantitation of an oral disease biomarker in human saliva by using a monolithic disposable cartridge designed to operate in a compact analytical instrument. Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. Using 20 microl of saliva, we demonstrate rapid (<10 min) measurement of the collagen-cleaving enzyme matrix metalloproteinase-8 (MMP-8) in saliva from healthy and periodontally diseased subjects. In addition to physiologically measurable indicators of periodontal disease, conventional measurements of salivary MMP-8 were used to validate the microfluidic assays described in this proof-of-principle study. The microchip-based POC diagnostic demonstrated is applicable to rapid, reliable measurement of proteinaceous disease biomarkers in biological fluids.

Integrated Microfluidic Platform for Oral Diagnostics

While many point-of-care (POC) diagnostic methods have been developed for blood-borne analytes, development of saliva-based POC diagnostics is in its infancy. We have developed a portable microfluidic device for detection of potential biomarkers of periodontal disease in saliva. The device performs rapid microfluidic chip-based immunoassays (<3-10 min) with low sample volume requirements (10 microL) and appreciable sensitivity (nM-pM). Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. The microfluidic chip has been integrated with miniaturized electronics, optical elements, such as diode lasers, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device and methods are being tested by detecting potential biomarkers in saliva samples from patients diagnosed with periodontal disease. Our microchip-based analysis can readily be extended to detection of biomarkers of other diseases, both oral and systemic, in saliva and other oral fluids.

Single-step enantioselective amino acid flux analysis by capillary electrophoresis using on-line sample preconcentration with chemical derivatization

Capillary electrophoresis (CE) represents a versatile platform for integrating sample pretreatment with chemical analysis because of its ability to tune analyte electromigration and band dispersion properties in discontinuous electrolyte systems. In this article, a single-step method that combines on-line sample preconcentration with in-capillary chemical derivatization is developed for rapid, sensitive, and enantioselective analysis of micromolar levels of amino acids that lack intrinsic chromophores by CE with UV detection. Time-resolved electrophoretic studies revealed two distinct stages of amino acid band narrowing within the original long sample injection plug occurring both prior to and after in-capillary labeling via zone passing by ortho-phthalaldehyde/ N-acetyl l-cysteine (OPA/NAC). This technique enabled direct analysis of d-amino acids in a 95% enantiomeric excess mixture with sub-micromolar detection limits and minimal sample handling, where the capillary functions as a preconcentrator, microreactor, and chiral selector. On-line sample preconcentration with chemical derivatization CE (SPCD–CE) was applied to study the enantioselective amino acid flux in Escherichia coli bacteria cultures, which demonstrated a unique l-Ala efflux into the extracellular medium. New strategies for high-throughput analyses of low-abundance metabolites are important for understanding fundamental physiological processes in bacteria required for screening the efficacy of new classes of antibiotics as well as altered metabolism in genetically modified mutant strains.

On-line sample preconcentration with chemical derivatization of bacterial biomarkers by capillary electrophoresis: A dual strategy for integrating sample pretreatment with chemical analysis

Simple, selective yet sensitive methods to quantify low-abundance bacterial biomarkers derived from complex samples are required in clinical, biological, and environmental applications. In this report, a new strategy to integrate sample pretreatment with chemical analysis is investigated using on-line preconcentration with chemical derivatization by CE and UV detection. Single-step enantioselective analysis of muramic acid (MA) and diaminopimelic acid (DAP) was achieved by CE via sample enrichment by dynamic pH junction with ortho-phthalaldehyde/N-acetyl-L-cysteine labeling directly in-capillary. The optimized method resulted in up to a 100-fold enhancement in concentration sensitivity compared to conventional off-line derivatization procedures. The method was also applied toward the detection of micromolar levels of MA and DAP excreted in the extracellular medium of Escherichia coli bacterial cell cultures. On-line preconcentration with chemical derivatization by CE represents a unique approach for conducting rapid, sensitive, and high-throughput analyses of other classes of amino acid and amino sugar metabolites with reduced sample handling, where the capillary functions simultaneously as a concentrator, microreactor, and chiral selector.