Determination of lysophosphatidylcholine using peroxidase-mimic PVP/PtRu nanozyme.

Lysophosphatidylcholine (LPC) can be used as a biomarker for diseases such as cancer, diabetes, atherosclerosis, and sepsis. In this study, we demonstrated the ability of nanozymes to displace the natural derived enzyme in enzyme-based assays for the measurement of LPC. Synthesized polyvinylpyrrolidone-stabilized platinum-ruthenium nanozymes (PVP/PtRu NZs) had a uniform size of 2.48 ± 0.24 nm and superb peroxidase-mimicking activity. We demonstrated that the nanozymes had high activity over a wide pH and temperature range and high stability after long-term storage. The LPC concentration could be accurately analyzed through the absorbance and fluorescence signals generated by the peroxidation reaction using the synthesized nanozyme with substrates such as 3,3',5,5'-tetramethylbenzidine (TMB) and 10-acetyl-3,7-dihydroxyphenoxazine (Ampliflu™ Red). LPC at a concentration of 0-400 µM was used for the analysis, and the coefficient of determination (R2) was 0.977, and the limit of detection (LOD) was 23.1 µM by colorimetric assay. In the fluorometric assay, the R2 was 0.999, and the LOD was 8.97 µM. The spiked recovery values for the determination of LPC concentration in human serum samples were 102-115%. Based on these results, we declared that PVP/PtRu NZs had an ability comparable to that of the native enzyme horseradish peroxidase (HRP) in the enzyme-based LPC detection method.

Mesoporous platinum nanoparticles as a peroxidase mimic for the highly sensitive determination of C-reactive protein.

The generation of a mesoporous structure in platinum nanoparticles can effectively enhance physical and chemical properties. In this study, mesoporous platinum nanoparticles (MPNs) were synthesized by a soft template-mediated one-pot chemical method. To develop a mesoporous structure, Pluronic F-127 was employed. The Pluronic F-127 surfactant forms self-assembled micelles, and the micelles act as the pore-directing agents in the synthesis of nanoparticles. Scanning electron microscopy results revealed that the MPN had a uniform size of 70 nm on average and a distinct mesoporous structure. The development of a concave mesoporous structure on the surface of the MPNs can increase the surface area and facilitate the efficient transport of reactants. The synthesized MPNs exhibited peroxidase-like activity. Furthermore, the MPNs showed excellent catalytic efficiency compared to HRP, due to the high surface area derived from the presence of the mesoporous structure. The peroxidase-like MPNs were applied to the enzyme-linked immunosorbent assay (ELISA) of C-reactive protein (CRP). The MPN-based ELISA exhibited sensitive CRP detection in the range from 0.24 to 7.8 ng/mL with a detection limit of 0.13 ng/mL. Moreover, the recoveries of the CRP concentrations in spiked human serum were 98.6% and 102%. These results demonstrate that as a peroxidase mimic, the MPNs can replace the natural enzymes in conventional ELISA for sensitive CRP detection.

Functionalized ultra-fine bimetallic PtRu alloy nanoparticle with high peroxidase-mimicking activity for rapid and sensitive colorimetric quantification of C-reactive protein.

The in situ synthesis is reported of citric acid-functionalized ultra-fine bimetallic PtRu alloy nanoparticles (CA@PtRu ANPs) through a simple one-pot wet chemical method. The cost-efficient CA@PtRu ANPs with an average diameter of 3.2 nm revealed to have enhanced surface area, peroxidase-like activity, high stability, and adequate availability of functional groups to bind biomolecules. Along with nanoparticle surface area, the surface charge has also significantly affected the peroxidase-like activity and the colloidal suspension stability. As an excellent immobilization matrix and peroxidase mimic, the CA@PtRu ANPs were utilized to develop non-enzymatic colorimetric immunoassay for rapid, selective, and sensitive quantification of C-reactive protein (CRP) biomarkers. In this immunoassay, CA@PtRu ANPs serve as enzyme mimic that significantly amplifies the color signals, and amine-functionalized silica-coated magnetic microbeads (APTES/SiO2@Fe3O4) act as CRP-recognizing capture probes. The absorbance curves of colorimetric immunoassay were measured in wavelengths between 550 and 750 nm, and the maximum absorbance at 652 nm was used to establish a linear relationship between absorbance and CRP concentrations. The developed colorimetric immunoassay showed rapid and sensitive quantification of CRP levels from 0.01 to 180 µg mL-1 with a LOD of 0.01 µg mL-1. Moreover, the mean recovery of CRP from spiked human serum samples lies between 97 and 109% (n = 3), which indicates that the proposed nanozyme-linked immunoassay has the potential to be used in rapid point-of-care applications.

Au nanoparticle-hydrogel nanozyme-based colorimetric detection for on-site monitoring of mercury in river water.

A sensitive on-site mercury sensing platform was developed for simple and effective monitoring of mercury levels in the field. The simple and practical mercury detection system was designed by integrating an Au nanoparticle-PEG hydrogel block nanozyme (Au-HBNz) into a polymer film-based colorimetric device. Upon addition of Hg2+ ions, Au-HBNz exhibited excellent peroxidase-like activity, catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine into a blue-colored product, which has a maximum absorbance at 652 nm. The resulting color intensity change was evaluated using a smartphone for simple and rapid Hg2+ detection with a broad detection range (0.008-20 µg∙mL-1) and a linear concentration-response relationship (R2 = 0.96). The detection limit (1.10 ng∙mL-1) was lower than the maximum permissible Hg2+ levels in drinking water set by the World Health Organization (6 ng∙mL-1) and U.S. Environmental Protection Agency (2 ng∙mL-1). The recoveries of Hg2+ determination in river water by spiking Hg2+ samples ranged from 92 to 106%, which indicated high validity and applicability of the Hg2+ detection system for field measurements. Thus, the developed sensor enables highly selective and efficient real-time monitoring of Hg2+.

Large-scale plasma patterning of transparent graphene electrode on flexible substrates.

Graphene, a two-dimensional carbon material, has attracted significant interest for applications in flexible electronics as an alternative transparent electrode to indium tin oxide. However, it still remains a challenge to develop a simple, reproducible, and controllable fabrication technique for producing homogeneous large-scale graphene films and creating uniform patterns with desired shapes at defined positions. Here, we present a simple route to scalable fabrication of flexible transparent graphene electrodes using an oxygen plasma etching technique in a capacitively coupled plasma (CCP) system. Ascorbic acid-assisted chemical reduction enables the large-scale production of graphene with solution-based processability. Oxygen plasma in the CCP system facilitates the reproducible patterning of graphene electrodes, which allows controllable feature sizes and shapes on flexible plastic substrates. The resulting graphene electrode exhibits a high conductivity of 80 S cm(-1) and a transparency of 76% and retains excellent flexibility upon hard bending at an angle of ±175° and after repeated bending cycles. A simple LED circuit integrated on the patterned graphene film demonstrates the feasibility of graphene electrodes for use in flexible transparent electrodes.

A single-walled carbon nanotube thin film-based pH-sensing microfluidic chip.

A novel microfluidic pH-sensing chip was developed based on pH-sensitive single-walled carbon nanotubes (SWCNTs). In this study, the SWCNT thin film acted both as an electrode and a pH-sensitive membrane. The potentiometric pH response was observed by electronic structure changes in the semiconducting SWCNTs in response to the pH level. In a microfluidic chip consisting of a SWCNT pH-sensing working electrode and an Ag/AgCl reference electrode, the calibration plot exhibited promising pH-sensing performance with an ideal Nernstian response of 59.71 mV pH(-1) between pH 3 and 11 (standard deviation of the sensitivity is 1.5 mV pH(-1), R(2) = 0.985). Moreover, the SWCNT electrode in the microfluidic device showed no significant variation at any pH value in the range of the flow rate between 0.1 and 15 µl min(-1). The selectivity coefficients of the SWCNT electrode revealed good selectivity against common interfering ions.

Dual-mode colorimetric and photothermal aptasensor for detection of kanamycin using flocculent platinum nanoparticles.

Chitosan (CS)-stabilized platinum nanoparticles (CS/PtNPs) were employed to develop a novel aptamer-based dual-mode colorimetric and photothermal biosensor for selective detection of kanamycin (KAN). As a peroxidase-like catalyst, the CS/PtNPs showed outstanding catalytic activity for the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). As a stabilizing agent, CS excelled at fixing the KAN binding aptamer on the surface of the CS/PtNPs, amplifying their catalytic activity and enhancing colloidal dispersion and stability. The oxidized TMB (TMBox) functioned as a signal for the colorimetric, photothermal aptasensor because of its observable absorbance of light in the visible and near-infrared (NIR) regions. When light from a NIR laser was absorbed by the TMBox in the reaction solution, heat was generated in inverse proportion to the KAN concentration. The developed colorimetric and photothermal modes of the aptasensor showed a linear detection range of 0.1-50 and 0.5-50 µM, with a limit of detection (LOD) of 0.04 and 0.41 µM, respectively. Moreover, the aptasensor successfully determined KAN concentrations in spiked milk samples, verifying the reliability and reproducibility in practical applications. The dual-mode aptasensor based on CS/PtNPs for KAN detection, utilizing both color change and heat generation signals through a single probe (TMBox), demonstrates rapid response, simplicity in operation, cost-effectiveness, and high sensitivity. In addition, unlike typical immunoassays, this aptamer-based peroxidase-like nanozyme activation and inhibition strategy required no washing process, which was very effective in terms of reducing the time required for an assay and sustaining a high sensitivity.

Chromo-Fluorogenic Rhodamine-Based Amphiphilic Probe as a Selective and Sensitive Sensor for Intracellular Cu(I) in Living Cells.

Fluorescent probes are widely studied for metal ion detection because of their multiple favorable properties such as high sensitivity and selectivity, quick response, naked eye detection, and in situ monitoring. However, optical probes that can effectively detect the Cu(I) level in cell interiors are rare due to the difficulty associated with selectively and sensitively detecting this metal ion in a cell environment. Therefore, we designed and synthesized three water-soluble probes (1-3) with a 1,3,5-triazine core decorated by three substituents: a hydrophobic alkyl chain, a hydrophilic maltose, and a rhodamine B hydrazine fluorophore. Among the probes, probe 1, which has an octyl chain and a branched maltose group, was the most effective at sensing Cu+ in aqueous solution. Upon addition of Cu+, this probe showed a dramatic color change from colorless to pink in daylight and displayed an intense yellow fluorescence emission under 365 nm light. The limit of detection and dissociation constant (Kd) of this probe were 20 nM and 1.1 × 10-12 M, respectively, which are the lowest values reported to date. The two metal ion-binding sites and the aggregation-induced emission enhancement effect, endowed by the branched maltose group and the octyl chain, respectively, are responsible for the high sensitivity and selectivity of this probe for Cu+ detection, as demonstrated by 1H NMR, dynamic light scattering, and transmission electron microscopy studies. Furthermore, the probe successfully differentiated the Cu(I) level of cancer cells from that of the normal cells. Thus, the probe holds potential for real-time monitoring of Cu(I) level in biological samples and bioimaging of cancer cells.

The therapeutic efficacy of silver loaded rhenium disulfide nanoparticles as a photothermal agent for cancer eradication.

Cancer is a life-threatening disease when it is diagnosed at a late stage or treatment procedures fail. Inhibiting cancer cells in the tumor environment is a significant challenge for anticancer therapy. The photothermal effects of nanomaterials are being studied as a new cancer treatment. In this work, rhenium disulfide (ReS2) nanosheets were made by liquid exfoliation with gum arabic (GA) and coated with silver nanoparticles (AgNPs) to produce reactive oxygen species that destroy cancer cells. The synthesized AgNP-GA-ReS2 NPs were characterized using UV, DLS, SEM, TEM, and photothermal studies. According to the DLS findings, the NPs were about 216 nm in size and had a zeta potential of 76 mV. The TEM and SEM analyses revealed that the GA-ReS2 formed single-layered nanosheets on which the AgNPs were distributed. The photothermal effects of the AgNP-GA-ReS2 NPs at 50 µg/mL were tested with an 808 nm laser at 1.2 W cm-2, and they reached 55.8 °C after 5 min of laser irradiation. MBA-MB-231 cells were used to test the cytotoxicity of the newly designed AgNP-GA-ReS2 NPs with and without laser irradiation for 5 min. At 50 µg/mL, the AgNP-GA-ReS2 showed cytotoxicity, which was confirmed with calcein and EtBr staining. The DCFH-DA and flow cytometry analyses demonstrated that AgNP-GA-ReS2 nanosheets under NIR irradiation generated ROS with high anticancer activity, in addition to the photothermal effects.

Apta-sensor for selective determination of dopamine using chitosan-stabilized Prussian blue nanoparticles.

Chitosan-stabilized Prussian blue nanoparticles (CS/PBNPs) were fabricated by a simple synthetic method and used to develop a novel aptamer-based colorimetric assay for selective determination of dopamine (DA). Scanning electron microscopy (SEM) images exhibited a uniform shape of the CS/PBNPs with an average diameter of 37.0 ± 3.2 nm. The CS/PBNPs exhibited strong peroxidase-like activity that catalyzed the reaction between 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). Chitosan was used for stabilization of the PBNPs and fixation of the DA aptamer on the surface of the CS/PBNPs. The catalytic mechanism of the CS/PBNPs was confirmed to involve first the decomposition of H2O2 into a hydroxyl radical (˙OH) and then oxidation of TMB by the ˙OH to produce a blue color. An aptamer-based colorimetric assay was made with the CS/PBNPs to detect DA at concentrations of 0.25-100 µM with a limit of detection (LOD) of 0.16 µM. For comparison, a gold nanoparticle (AuNP)-based apta-sensor detected DA in concentrations of 1-25 µM with a LOD of 0.55 µM. The recovery results of DA concentrations (0.25, 0.5, and 1 µM) from spiked human serum were 92.6%, 102.1%, and 103.9%, verifying the reliability and reproducibility of the CS/PBNP-based apta-sensor for determination of DA level in clinical applications. Moreover, compared to traditional immunoassay, this aptamer-based nanozyme activation/inhibition system needs no washing step, which is very useful to shorten the assay time and maintain high sensitivity.

Ratiometric orange fluorescent and colorimetric highly sensitive imidazolium-bearing naphthoquinolinedione-based probes for CN- sensing in aqueous solutions and bio-samples.

The widespread use of cyanide (CN-) in industry results in contamination of various effluents such as drain, lake, and tap water, an imminent danger to the environment and human health. We prepared naphthoquinolinedione (cyclized; 1-5) and anthracenedione (un-cyclized) probes (6-7) for selective detection of CN-. The addition of CN- to the probe solutions (1-5) resulted in a color change from pale green to orange under 365 nm illumination. The nucleophilic addition of CN- to C2 of the imidazolium ring of the probes is responsible for selective CN- detection. Among all probes, 1 gave the lowest fluorescence-based LOD of 0.13 pM. In contrast, the un-cyclized probes (6 and 7) were substantially inferior to the cyclized counterparts (1 and 2, respectively) for detecting a trace amount of CN-. The notably low LOD displayed by probe 1 was maintained in the detection of CN- in real food samples, human fluids, and human brain cells. This is the first report studying imidazolium-bearing naphthoquinolinedione-based probes for CN- sensing in 100% water.

Multicomponent metal-organic framework nanocomposites for tumor-responsive synergistic therapy.

Targeted tumor therapy through tumor microenvironment (TME)-responsive nanoplatforms is an emerging treatment strategy used to enhance tumor-specificity to selectively kill cancer cells. Here, we introduce a nanosized zeolitic imidazolate framework-8 (ZIF-8) that simultaneously contains natural glucose oxidase (GOx) and Prussian blue nanoparticles (PBNPs) to construct multi-component metal-organic framework nanocomposites (denoted as ZIF@GOx@PBNPs), which possess cascade catalytic activity selectively within the TME. Once reaching a tumor site, GOx and PBNPs inside the nanocomposites are sequentially released and participate in the cascade catalytic reaction. In weak acidic TME, GOx, which effectively catalyzes the oxidation of intratumoral glucose to hydrogen peroxide (H2O2) and gluconic acid, not only initiates starvation therapy by cutting off the nutrition source for cancer cells but also produces the reactant for sequential Fenton reaction for chemodynamic therapy. Meanwhile, PBNPs, which are released from the ZIF-8 framework dissociated by acidified pH due to the produced gluconic acid, convert the generated H2O2 into harmful radicals to melanomas. In this way, the cascade catalytic reactions of ZIF@GOx@PBNPs enhance reactive oxygen species production and cause oxidative damage to DNA in cancer cells, resulting in remarkable inhibition of tumor growth. The tumor specificity is endowed by using the biomolecules overexpressed in TME as a "switch" to initiate the first catalytic reaction by GOx. Given the significant antitumor efficiency both in vitro and in vivo, ZIF@GOx@PBNPs could be applied as a promising therapeutic platform enabling starvation/chemodynamic synergism, high therapeutic efficiency, and minimal side effects.

Gum Arabic-mediated liquid exfoliation of transition metal dichalcogenides as photothermic anti-breast cancer candidates.

Transition metal dichalcogenides (TMDs) have gained considerable attention for a broad range of applications, including cancer therapy. Production of TMD nanosheets using liquid exfoliation provides an inexpensive and facile route to achieve high yields. In this study, we developed TMD nanosheets using gum arabic as an exfoliating and stabilizing agent. Different types of TMDs, including MoS2, WS2, MoSe2, and WSe2 nanosheets, were produced using gum arabic and were characterized physicochemically. The developed gum arabic TMD nanosheets exhibited a remarkable photothermal absorption capacity in the near-infrared (NIR) region (808 nm and 1 W⋅cm-2). The drug doxorubicin was loaded on the gum arabic-MoSe2 nanosheets (Dox-G-MoSe2), and the anticancer activity was evaluated using MDA-MB-231 cells and a water-soluble tetrazolium salt (WST-1) assay, live and dead cell assays, and flow cytometry. Dox-G-MoSe2 significantly inhibited MDA-MB-231 cancer cell proliferation under the illumination of an NIR laser at 808 nm. These results indicate that Dox-G-MoSe2 is a potentially valuable biomaterial for breast cancer therapy.

Simultaneous detection of ultratrace lead and copper with gold nanoparticles patterned on carbon nanotube thin film.

Highly sensitive detection of a Pb(2+)-Cu(2+) mixture using gold nanoparticles patterned on single-walled carbon nanotube (AuNP-SWCNT) film is reported. The gold nanoparticles were deposited electrochemically on carbon nanotube film using a cyclic voltammetry technique. The film showed a homogeneous size and density that could be easily controlled by the potential scanning cycle and gold precursor concentration. Square wave stripping voltammetry (SWSV) was applied to the simultaneous detection of Pb(2+) and Cu(2+) under optimized conditions. The AuNP-SWCNT electrode exhibited a high increase in sensitivity with a limit of detection of 0.546 ppb (R(2) = 0.984) and 0.613 ppb (R(2) = 0.991) for Pb(2+) and Cu(2+) ions, respectively, in a mixture of Pb(2+)-Cu(2+) solution (S/N = 3, n = 5), and a good linear response in the range from 3.31 ppb to 22.29 ppb. The electrode exhibited high reproducibility in repetitive measurements with a relative standard deviation as low as 4.2% and 2.6% for Pb(2+) and Cu(2+) ions, respectively. An interference study showed that Sb(3+), As(3+), Zn(2+), Ca(2+), and Na(+) ions did not have a significant effect. This study demonstrated an alternative approach to the rapid and reliable detection of heavy metals of environmental interest.

Fucoidan-based nanoparticles: Preparations and applications.

Nanoparticle-based therapy has gained much attention in the pharmaceutical industry. Fucoidan is a sulfated polysaccharide naturally derived from marine brown algae and is widely used for medical applications. We explore preparation of fucoidan-based nanoparticles and their biomedical applications in the current review. The fucoidan-based nanoparticles have been synthesized using microwave, emulsion, solvent evaporation, green synthesis, polyelectrolyte self-assembly, precipitation, and ultrasonication methods. The synthesized nanoparticles have particle sizes ranging from 100 to 400 nm. Therefore, fucoidan-based nanoparticles have a variety of potential therapeutic applications, including drug delivery, cancer therapies, tissue engineering, antimicrobial applications, magnetic resonance imaging contrast, and atherothrombosis imaging. For example, fucoidan nanoparticles have been used to deliver curcumin, dextran, gentamicin, epigallocatechin gallate, and cisplatin for cancer therapies. Furthermore, fucoidan nanoparticles coupled with metal nanoparticles have been used to target and recognize clinical conditions for diagnostic purposes. Hence, fucoidan-based nanoparticles have been helpful for biomedical applications.

Alginate-based Composite Microspheres: Preparations and Applications for Bone Tissue Engineering.

Alginate-based biomaterials have been extensively studied for bone tissue engineering. Scaffolds, microspheres, and hydrogels can be developed using alginate, which is biocompatible, biodegradable, and able to deliver growth factors and drugs. Alginate microspheres can be produced using crosslinking, microfluidic, three-dimensional printing, extrusion, and emulsion methods. The sizes of the alginate microspheres range from 10 µm to 4 mm. This review describes the chemical characterization and mechanical assessment of alginatebased microspheres. Combinations of alginate with hydroxyapatite, chitosan, collagen, polylactic acid, polycaprolactone, and bioglass were discussed for bone tissue repair and regeneration. In addition, alginate combinations with bone morphogenetic proteins, vascular endothelial growth factor, transforming growth factor beta- 3, other growth factors, cells, proteins, drugs, and osteoinductive drugs were analyzed for tissue engineering applications. Furthermore, the biocompatibility of developed alginate microspheres was discussed for different cell lines. Finally, alginate microsphere-based composites with stem cell interaction for bone tissue regeneration were presented. In the present review, we have assessed the preclinical research on in vivo models of alginatebased microspheres for bone tissue repair and regeneration. Overall, alginate-based microspheres are potential candidates for graft substitutes and the treatment of various bone-related diseases.

Biomimetic chitosan with biocomposite nanomaterials for bone tissue repair and regeneration.

Biomimetic materials for better bone graft substitutes are a thrust area of research among researchers and clinicians. Autografts, allografts, and synthetic grafts are often utilized to repair and regenerate bone defects. Autografts are still considered the gold-standard method/material to treat bone-related issues with satisfactory outcomes. It is important that the material used for bone tissue repair is simultaneously osteoconductive, osteoinductive, and osteogenic. To overcome this problem, researchers have tried several ways to develop different materials using chitosan-based nanocomposites of silver, copper, gold, zinc oxide, titanium oxide, carbon nanotubes, graphene oxide, and biosilica. The combination of materials helps in the expression of ideal bone formation genes of alkaline phosphatase, bone morphogenic protein, runt-related transcription factor-2, bone sialoprotein, and osteocalcin. In vitro and in vivo studies highlight the scientific findings of antibacterial activity, tissue integration, stiffness, mechanical strength, and degradation behaviour of composite materials for tissue engineering applications.

Remineralizing Potential of Natural Nano-Hydroxyapatite Obtained from Epinephelus chlorostigma in Artificially Induced Early Enamel Lesion: An In Vitro Study.

Dental caries is a common problem in adolescents, leading to permanent loss of teeth or cavitation. Caries is a continuous process wherein demineralization and remineralization occur regularly. Hydroxyapatite (HA) is one of the most biocompatible and bioactive materials, as it closely resembles the mineral composition of teeth. The present study deals with isolating hydroxyapatite from fish bone (Epinephelus chlorostigma) by alkaline hydrolysis and thermal calcination. The isolated nano HA was characterized using FT-IR, XRD, TGA, FE-SEM-EDX, and HR-TEM analysis. The nano HA isolated by alkaline hydrolysis is nontoxic, and the cells are viable. The isolated HA enhances the proliferation of L929 cells. The remineralization potential of the extracted nano HA was evaluated in healthy premolars by DIAGNOdent/laser fluorescence quantification, surface microhardness test, and SEM-EDX analysis. Surface morphological observations in SEM and EDX analyses show that thermally calcined HA and alkali-treated HA can induce mineralization and deposit minerals. Therefore, HA obtained from Epinephelus chlorostigma could be a potential biomaterial for treating early caries.

Enzyme kinetic measurements using a droplet-based microfluidic system with a concentration gradient.

In this paper, we propose a microfluidic device that is capable of generating a concentration gradient followed by parallel droplet formation within channels with a simple T-junction geometry. Linear concentration gradient profiles can be obtained based on fluid diffusion under laminar flow. Optimized conditions for generating a linear concentration gradient and parallel droplet formation were investigated using fluorescent dye. The concentration gradient profile under diffusive mixing was dominated by the flow rate at sample inlets, while parallel droplet formation was affected by the channel geometry at both the inlet and outlet. The microfluidic device was experimentally characterized using optimal layout and operating conditions selected through a design process. Furthermore, in situ enzyme kinetic measurements of the ß-galactosidase-catalyzed hydrolysis of resorufin-ß-d-galactopyranoside were performed to demonstrate the application potential of our simple, time-effective, and low sample volume microfluidic device. We expect that, in addition to enzyme kinetics, drug screening and clinical diagnostic tests can be rapidly and accurately performed using this droplet-based microfluidic system.