Reagent Volumes Research Articles

A Miniaturized Optofluidic Glucose Monitoring System Based on Enzyme Colorimetry

Continuous glucose monitoring is of great significance in the diagnosis and treatment of diabetes. In this paper, a miniaturized and automatic optofluidic system was designed for continuous glucose monitoring. Interstitial fluid (ISF) was first obtained by an ISF transdermal extracting chip. Then, the glucose concentration in the ISF was detected by enzyme colorimetry. For accurate detection of the glucose concentration, the volume of transdermally extracted ISF was measured, and the volumes of ISF and colorimetric reagent in the mixed sample were controlled and mixed uniformly. To test the precision and stability of the system, different concentrations of glucose solutions (30–200 mg/dL) were used to simulate ISF extracted from skin. The results revealed that the measured glucose concentration correlated well with the reference concentration (R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> = 0.9976). In addition, the system achieved high-precision measurement of a low glucose concentration (30 mg/dL) even as the ISF volume fluctuated widely (0.5– <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$2.5 ~\mu \text{L}$ </tex-math></inline-formula> ), and the average absolute error was only 1.27%. Therefore, the system realized high precision for automatic and continuous monitoring of glucose in microvolume ISF samples, which would be a promising tool for the continuous monitoring of blood glucose concentration.

Advancements in Ionomers for Fuel Cell Ion Exchange Membranes, Lithium Ion Battery Electrolyte Membranes and Supercapacitors

Scientists are being pushed to find more sustainable energy conversion and storage solutions as pollution levels rise, oil costs rise, and climate change becomes more problematic. Devices such as fuel cells, redox flow batteries, and electrolyzers are examples of devices that may significantly cut greenhouse gas emissions. These devices rely on ionic conductive polymers or ionomers (protonic, anionic, and amphoteric). For a variety of applications ranging from mobile to automotive and cogeneration systems, such ionomers must have high chemical and mechanical stability, performance and durability, low reagent permeability, and weight, volume, and current density. Unfortunately, the expensive cost of perfluorinated ionomers, as well as anionic polymers' low stability in alkaline settings, limit their usage.

Numerical Modelling of Mixing in a Microfluidic Droplet Using a Two-Phase Moving Frame of Reference Approach.

Droplets generated in microfluidic channels are effective self-contained micromixers and micro-reactors for use in a multiplicity of chemical synthesis and bioanalytical applications. Droplet microfluidic systems have the ability to generate multitudes of droplets with well-defined reagent volumes and narrow size distributions, providing a means for the replication of mixing within each droplet and thus the scaling of processes. Numerical modelling using computational fluid dynamics (CFD) is a useful technique for analysing and understanding the internal mixing in microfluidic droplets. We present and demonstrate a CFD method for modelling and simulating mixing between two species within a droplet travelling in straight microchannel, using a two-phase moving frame of reference approach. Finite element and level set methods were utilised to solve the equations governing the coupled physics between two-phase flow and mass transport of the chemical species. This approach had not been previously demonstrated for the problem of mixing in droplet microfluidics and requires less computational resources compared to the conventional fixed frame of reference approach. The key conclusions of this work are: (1) a limitation of this method exists for flow conditions where the droplet mobility approaches unity, due to the moving wall boundary condition, which results in an untenable solution under those conditions; (2) the efficiency of the mixing declines as the length of the droplet or plug increases; (3) the initial orientation of the droplet influences the mixing and the transverse orientation provides better mixing performance than the axial orientation and; (4) the recirculation inside the droplet depends on the superficial velocity and the viscosity ratio.

Simplified Method for the Determination of Total Kjeldahl Nitrogen in Wastewater

The increase in total Kjeldahl nitrogen (TKN) concentrations is correlated with increases in cyanobacterial bloom biomass. Standard methods for the measurement of TKN are tedious, costly, time-consuming and involve the use of hazardous catalysts, such as mercury, high temperatures and significant amounts of toxic acids and bases. Since TKN plays a pivotal role in influencing algal blooms, there is an urgent need to develop simpler, safer and more accurate methods for the determination of TKN. The simplified TKN method (s-TKN™) developed by Hach® offers several advantages over the traditional TKN method, including eliminating the use of mercury, requiring low sample and reagent volumes and being cost-efficient and user-friendly. This communication presents preliminary results comparing the efficacy of s-TKN™ and the standard method, using commonly used primary standards and waste, estuarine and lake water matrices. For all primary standards analyzed, the s-TKN™ method exhibited good accuracy across a wide range of concentrations. The repeatability for the glycine–para-toluene sulfonic acid (Gly-PTSA) standard using the s-TKN™ method was 4.1% at the highest concentrations analyzed, with overall repeatability across concentrations comparable to the standard EPA method. For wastewater, estuarine and lake matrices, a good correlation (r2 = 0.9917) between the two methods and no statistical difference in the values (p &gt; 0.05) were obtained between two methods. Preliminary studies indicate that the s-TKN™ method has the potential to reduce the expenditure associated with the cost of analysis and has the potential to be a safer and cheaper alternative, while providing comparable analytical results to the standard method.

Green Analytical Approach for the Determination of Zinc in Pharmaceutical Product Using Natural Reagent.

A selective, sensitive, and environmentally safe spectrophotometry method was developed and validated for the determination of zinc in pharmaceutical substances using natural reagents obtained from the leaves of plant Acacia catechu. Different factors were optimized such as volume of reagent, selection of pH, and stability of the color complex. The drug showed a stable yellowish orange color complex at 550 nm. The greenness of the methods was estimated using an eco-scale tool where the presented method was found to be excellent green with an ecoscore of 84 based on spectrophotometric determination. Also, the greenness of the method was assessed by the Green Analytical Procedure Index and found to be eco-friendly. The method was validated in conformance with ICH guidelines, with acceptable values for linearity, accuracy, precision, LOD, and LOQ. The linearity range for zinc sulphate was 5–25 μg mL−1 with an R2 value of 0.996. The % RSD for intraday precision and interday precision was less than 2%. The suggested method can be employed for the economic analysis of zinc in its pure form and various formulations. The presented spectrophotometric method is the first analytical method for the analysis of zinc present in zinc sulphate and showed greater ecoscale as compared to the official method.

Testing the Ion AmpliSeq™ HID Y-SNP Research Panel v1 for performance and resolution in admixed South Americans of haplogroup Q

Y haplogroups, defined by Y-SNPs, allow the reconstruction of the human Y chromosome genealogy, which is important for population, evolutionary and forensic genetics. In this study, Y-SNPs were typed and haplogroups inferred with the MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1, as a high-throughput approach. Firstly, the performance of the panel was evaluated with different DNA input amounts, reagent volumes and cycle numbers. DNA-inputs from 0.5 to 1 ng generated the most balanced read depth. Combined with full reagent and 19 cycles, this offered the highest number of amplicons with a sequencing read depth of at least 20 reads. Secondly, the sub-haplogroups of 182 admixed South Americans and Greenlanders belonging to haplogroup Q were inferred and tested for potential improvement in resolution. Most samples were assigned to lineage Q-M3 with some samples assigned to lineages upstream (Q-M346, L56, L57; Q-L331, L53; Q-L54; Q-CTS11969, CTS11970) or parallel (Q-L330, L334; Q-Z780/M971) to Q-M3. Only one sample was assigned to a downstream lineage (Q-Z35615, Z35616). Most individuals of haplogroup Q with NAM ancestry could neither be distinguished from each other, nor from half of the Greenlandic samples. Typing additional, known SNPs within lineage Q-M3, Z19483 and SA05, increased the resolution of predicted haplogroups. The search for novel variants in the sequenced regions allowed the detection of 42 variants and the subdivision of lineage Q-M3 into new subclades. The variants found in six of these subclades were exclusive to certain South American countries. In light of the limited differentiation of haplogroup Q samples, the additional information on known or novel SNPs disclosed in this study when using MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1 should be included in the Yleaf software, to increase the differentiation of lineage Q-M3.

A fully-printed electrochemical platform for assisted colorimetric detection of phosphate in saliva: Greenness and whiteness quantification by the AGREE and RGB tools

Herein, we report the environmental impact quantification of a newly developed fully printed electrochemical device to assist a colorimetric detection of phosphate in saliva. The evaluation of the analytical procedure was performed according to the principles of Green Analytical Chemistry and White Analytical Chemistry. The standard method for phosphate detection relies on a reaction between phosphate and molybdate in presence of antimony potassium tartrate and ascorbic acid, using strong acid conditions and high volumes of reagents (100–500 mL). To deliver an eco-friendly method, we have combined a screen-printed electrode with a liquid electrolyte battery and inkjet-printed conductive paths to develop a fully printed device on a flexible polymer substrate avoiding the use of ascorbic acid and using a small amount of reagents. The printed sensor was first developed and optimized for phosphate detection in saliva, allowing for a detection limit equal to 26 µM and satisfactory repeatability (relative standard deviation value of 7.5%). Finally, the AGREE and the RGB assessment tools were applied for a quantitative evaluation of the proposed sensor and reference method, in agreement with the Green Analytical and White Analytical principles. The results demonstrated the lower environmental impact of the proposed sensor, as well as the suitability of this novel approach for phosphate detection in saliva.

Computational study of an integrated microfluidic device for active separation of RBCs and cell lysis

Separation and lysis of RBCs play an important role in diagnosis of different diseases. Although they have been partially studied in several researches, a comprehensive study on integrating both separation and lysis units on a single chip has been seen rarely in the literature. Also, the factors related to the chemical lysis process have not been investigated in detail. In this study, we introduce a novel microfluidic channel design for sequential RBC's separation and lysis. For the separation part, an active method with an electric field was applied to the cells. Besides, a novel mixer was designed for mixing the cell solution and lysis reagent. In the lysis section, we used a mathematical model to simulate cell lysis. It is shown that in electric field with intensity of 2.7 V and frequency of 70 kHz, RBCs are separated with an efficiency of 100%. It is also shown that in using a serpentine mixed with barrier, the mixing efficiency rises to 89.35%. Also, the concentration and volume fraction of lysis reagent were investigated as effecting factors on the cell lysis's efficiency. Based on the results when lysis reagent concentration and volume fraction are 15% and 83.3%, respectively, RBCs are fully lysed.

DOE Assisted RP-HPLC Method Development and Validation for Estimation of L-Cysteine and Cystine with Dansyl Chloride derivatization in Presence of Amino Acid Mixture

The aim of the present study is to apply Design of Experiments (DoE), to develop an assay and optimize the derivatization reaction. DoE study type is Response Surface, Sub type is Split-plot, Design is D-optimal method was used in the study for the separation of L-Cysteine and L-Cystine in presence of amino acids by RP-HPLC. DoE Response Surface Split-plot method was used in this study. DoE allows to interpret the results with better outcome and enhanced understanding. Pre-column derivatizing agent Dansyl Chloride was used for dansylation of amino acids; 10mm Ammonium Acetate buffer PH 6.3, Acetonitrile was used as mobile phase in Eclipce XDB C18 column (150mm × 4.6mm, 5μm). Derivatizing reagent volume, derivatization reaction heating time, and temperature was evaluated as variables in the DoE. The wavelength for all amino acids is 222nm. Conclusively DoE as an efficient tool for optimization of dansylation reaction for the separation of L-Cysteine and L-Cystine in presence of amino acids. Method development was established and the design is validated. The proposed method has adequate reproducibility, specificity and accurate for the estimation of L-Cystine and L-Cystine in the presence of amino acids in routine analysis.

Fast DNA biosensing based on isothermal amplification, unmodified gold nanoparticles, and smartphone detection

The protection of the consumer against foodborne illnesses and fraudulent practices requires methods capable of detecting critical components. Classical instrumental and DNA-based technologies have assay time, portability, and cost limitations. Thus, food control needs alternative methodologies for massive and cost-effective screening. Herein, we report an instrument-free method based on detecting genes that encode proteins related to food allergies and ingredients associated with illegal practices. Tailed recombinase polymerase amplification (tailed-RPA) provided the selective isothermal amplification of target regions. A reproducible and fast optical detection was developed based on the salt-induced aggregation of 15 nm gold nanoparticles (AuNPs). The target presence was directly observed (naked-eye detection) and quantified using a smartphone and RGB image decomposition. The advantages arise from the effective formation of a hybrid complex between non-functionalized nanoparticles and amplification products with a single-strand tail, featuring short incubation time, simplicity, and low sample and reagent volumes. As proof of concept, two targets were determined: trnL gene and ITS region. The first sequence is located in the chloroplast genome of cereals and is valuable to control the adulteration of meat products. The second is a fragment common to the three main gluten-containing cereals, applicable to the indirect detection of this allergen. The novelty also is the integration of a low-cost assay platform and a straightforward interpretation system to foster its implementation in the industry. The RPA-AuNP assay offered a good sensitivity (genomic DNA 0.8 ng), selectivity (absence of unspecific response), reproducibility (standard deviation 2–11%), and accuracy in marketed food products (100%). Therefore, a competitive biosensing system enables better control according to food industry/consumers demands from Farm to Fork strategy.

An optimization of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid response teams.

Background:The COVID-19 pandemic relies on real-time polymerase chain reaction (qRT-PCR) for the detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to facilitate roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers' recommendations to sustain the testing capability in a resource-limited setting. Methods:We used a SARS-CoV-2 positive control RNA sample to generate several 10-fold dilution series that were used for optimization and comparison of the performance of the four qRT-PCR assays: i) Charité Berlin primer-probe set, ii) European Virus Archive - GLOBAL (EVAg) primer-probe set, iii) DAAN premixed commercial kit and iv) Beijing Genomics Institute (BGI) premixed commercial kit. We adjusted the manufacturer- and protocol-recommended reaction component volumes for these assays and assessed the impact on cycle threshold (Ct) values. Results:The Berlin and EVAg E gene and RdRp assays reported mean Ct values within range of each other across the different titrations and with less than 5% difference. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit improved in performance following a reduction of the reaction components. Conclusion:We achieved a 2.6-fold and 4-fold increase in the number of tests per kit for the commercial kits and the primer-probe sets, respectively. All the assays had optimal performance when the primers and probes were used at 0.375X, except for the Berlin N gene assay. The DAAN kit was a reliable assay for primary screening of SARS-CoV-2 whereas the BGI kit's performance was dependent on the volumes and concentrations of both the reaction buffer and enzyme mix. Our recommendation for SARS-CoV-2 diagnostic testing in resource-limited settings is to optimize the assays available to establish the lowest volume and suitable concentration of reagents required to produce valid results.

Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2

BackgroundThe COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost 1 million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs.ResultsWe present the ‘Mini-XT’ miniaturized tagmentation-based library preparation protocol available on protocols.io (https://doi.org/10.17504/protocols.io.bvntn5en). SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. The final version of Mini-XT has been used to sequence 4384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees.ConclusionsThe Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.

New insights for integration of nano particle with microfluidic systems for sensor applications

A biosensor is a compact device, which utilizes biological derived recognition component, immobilized on a transducer to analyze an analyte. Nanoparticles with their unique chemical and physical properties are versatile in their applications to develop as sensors. Different nanoparticles play different roles in the sensing systems like metal and metal oxide nanoparticles. The application of Gold, Silver and Copper nanoparticles will be discussed in brief. The nanoparticles typically function as substrates for immobilization of biomolecules, as catalytic agent, electron transfer agent between electrode surface and the biomolecules, and as reactants. Microfluidic deals with manipulating very small volumes of fluids (micro and nanoliters). This miniaturized platform enhances control of flow conditions and mixing rate of fluids. The microfluidics improves the sensitivity of the analysis, and reduces the volumes of sample and reagent in the analysis. The review specifically aims at representing microfluidics-based sensors and nanoparticle based sensors. This review will also focus on probable merger of these two fields to take advantage of both the fields and this will help in pushing the boundaries of these fields further more.

Ensuring the safety of chemical dosing

The article deals with the issues of ensuring the safe dosing of small volumes of chemical reagents with positive displacement pumps. The results of simulation of hydrodynamic processes in valve assemblies are presented. Studies have shown the low efficiency of the reagent supply process using ball and poppet valves. The use of hydraulic diaphragm and vortex diodes is proposed. Based on amathematical modeling, the design features of hydrodiodes and the parameters of their effective operation are substantiated, which makes it possible to exclude fluid pulsation and increase the flow rate. A diaphragm hydrodiode is suitable for a metering pump with a flow rate of at least 5 l/h. The vortex is capable of replacing ball and poppet valves at low and ultra-low flows, and it has been established that its diode resistance is more than 6, and its diode is more than 2 in terms of flow.

Characterization of pressure-driven water flows in nanofluidic channels by mass flowmetry.

With developments in analytical devices promoted by nanofluidics, estimation of the flow rate in a nanochannel has become important to calculate volumes of samples and reagents in chemical processing. However, measurement of the flow rate in nanospaces remains challenging. In the present study, a mass flowmetry system was developed, and the flow rate of water by pressure-driven flow in a fused-silica nanochannel was successfully measured in picoliters per second. We revealed that the water flow rate is dependent on the viscosity significantly increased in a square nanochannel with 102nm width and depth (3.6 times higher than the bulk viscosity for a representative channel size of 190nm) and slightly increased in a plate nanochannel with micrometer-scale width and 102nm depth (1.3 times higher for that of 234nm), because of dominant surface effects. The developed method and results obtained will greatly contribute to nanofluidics and other related fields.

Adjusting Acid Digestion Procedure to Determine the Concentration Levels of Essential Metals in Teff Samples

An efficient and cost effective method was developed and optimized for the digestion of Fe, Ca, Cu, Zn, Mn, Al, Cd and Pb in teff samples by using MP-AES. The effect of the digestion media, digestion time and digestion temperature on the efficiency of extraction of metals from the teff samples was examined. The levels of the metals in the Teff samples were determined for the samples collected from three selected areas (Bure, Debre Markos and Bahir Dar) of Ethiopia. Concentrated HNO₃ and HClO₄ were determined to be the most efficient digestion media for all the metals studied. After proper sample pretreatment, 5 mL HNO₃: 1 mL HClO₄, 240°C and 2:30 h were the optimized digestion conditions. Then using the optimized conditions sample preparation was made and levels of metals were determined by micro-plasma atomic absorption spectroscopy (MP-AES). The optimized conditions in this study were economical in terms of time and reagent volumes used compared to other authentic reports. The accuracy of the optimized procedure was evaluated by analyzing the digest of the spiked samples with standard solution and the percentage recoveries varied from 92% to 104%, which is good and was in the acceptable range of 90% ± 10.

Validation of Microchip Based RT-PCR ABC Test (InfA/B &amp;amp; COVID-19) in Clinical Samples

To contain the rapid and global spread of SARS-CoV-2, it is essential to develop an accurate and sensitive test system to address pandemic bottlenecks, simplified sample collection, and no sample prep. While meeting the demand of testing large populations, the miniaturized volume of assay reagents and offering rapid results is the need in such scenarios. Moreover, in view of the reports of co-infections and overlapping symptoms of influenza caused by Influenza A or Influenza B, and COVID-19 caused by SARS-CoV-2, a test system with three targets can be supportive for accurate clinical diagnosis. In this presentation, we evaluated the performance of a test comprising Microchip RT-PCR Influenza and COVID-19 Detection System for identifying these three viral pathogens in nasal swabs and saliva specimens. A rapid and simplified total nucleic acid extraction method was developed and validated for the reliable, high-throughput simultaneous detection of respiratory viruses causing Influenza (type A and type B viruses) and COVID-19 (SARS-CoV-2 virus) using the microchip-based AriaDNATM platform deriving the name ABC Test. The test system was evaluated using 81 nasal swab samples, 77 clinical saliva samples, 5 blind CAP reference samples, and RNA standards. The limit of detection (LoD) was assessed using SARS-CoV-2, Influenza A, and Influenza B RNA standards. The multiplex ABC Test microchip displayed LoD of 14 copies/μL for SARS-CoV-2 and approximately 26 copies/μL for influenza A, and 140 copies/μL for influenza B, respectively. The ABC Test offers rapid multiplex one-step RT-PCR in 32 minutes for 45 cycles as the miniaturized reaction of 1.2 μL offering a highly sensitive, robust, and accurate assay for the detection of influenza A/B, and SARS-CoV-2.

In situ synthesis of reagents in paper-based analytical devices using paper stacking.

This article demonstrates a technique for the in situ synthesis of an insoluble analytical reagent in paper analytical devices, using paper stacking. Previously, our group has demonstrated that stacking a fast-wicking paper membrane on top of a slow-wicking paper membrane containing dried reagents enables the uniform rehydration of the dried reagents over large areas. This technique is utilized here to fabricate distance-based sweat chloride quantification strips, which requires the synthesis of insoluble silver chromate as an analytical reagent in paper. The in situ generation of silver chromate for sweat chloride detection was previously accomplished by manually dipping a hydrophobically patterned paper channel into multiple precursor solutions with intermittent washing and drying. Compared to the previous technique, the stacking method obviates the need for (i) patterning hydrophobic barriers in paper for creation of flow channels, and (ii) multiple dipping steps that need large reagent volumes. The method is amenable to large scale manufacturing as the insoluble reagent can be synthesized uniformly over large paper areas and can then be cut into multiple sensing strips. The developed sensor has a limit of detection of ∼0.3 mM and a wide linear dynamic range of 0-120 mM for the detection of chloride ions, which enables the diagnosis of cystic fibrosis, characterized by sweat chloride levels greater than 60 mM. This simple technique of in situ synthesis of insoluble analytical reagents in paper could enable expanding the range of analytical chemistries that may be performed in paper-based analytical devices.

Droplet Magnetofluidic Assay Platform for Quantitative Methylation-Specific PCR.

Early cancer detection requires identification of cellular changes resulting from oncogenesis. Abnormal DNA methylation patterns occurring early in tumor development have been widely identified as early biomarkers for multiple types of cancer tumors. Methylation-Specific PCR (MSP) has permitted highly sensitive detection of these methylation changes at known biomarker locations. MSP requires multiple sample preparation steps including protein digestion, DNA isolation, and bisulfite conversion prior to detection. In this work, we present a streamlined assay platform and instrumentation for integration of all sample processing steps required to obtain quantitative MSP signal from raw biological samples through the use of droplet magnetofluidic principles. In conjunction with this platform, we present a streamlined protocol for solid-phase DNA extraction from cells and bisulfite conversion of genomic DNA, minimizing the processing steps and reagent volume for implementation on a compact assay platform.

Microcrater-Arrayed Chemiluminescence Cell Chip to Boost Anti-Cancer Drug Administration in Zebrafish Tumor Xenograft Model.

Simple SummaryPersonalized drug cocktails have been widely implemented in cancer treatment, due to their merits of using a drug synergistic combination rather than a single effector. Through the incorporation of a cell microarray chip, the usage of both cell amount and reagent can be optimized and effectively reduced in cost. Herein, we present a laser direct writing strategy to fabricate a microcrater-arrayed (µCA) chip incorporated with an automatic liquid handling platform. Each cell droplet with a critical volume of 200 nL containing 100 SK-N-DZ or MCF-7 cells was utilized. The drug synergy screening took less than 30 s for a total of 81 tests. The results show that the potent drug prediction of the µCA chip is more accurate than the conventional 96-well plate assay, which are all compared using zebrafish DiI-labelled tumor xenograft models. Taken together, these findings may impact high-throughput drug screening and personalized drug medicine.Purpose: The aim of this study was to develop a rapid and automatic drug screening platform using microcrater-arrayed (µCA) cell chips. Methods: The µCA chip was fabricated using a laser direct writing technique. The fabrication time required for one 9 × 9 microarray wax chip was as quick as 1 min. On a nanodroplet handling platform, the chip was pre-coated with anti-cancer drugs, including cyclophosphamide, cisplatin, doxorubicin, oncovin, etoposide, and 5-fluorouracil, and their associated mixtures. Cell droplets containing 100 SK-N-DZ or MCF-7 cells were then loaded onto the chip. Cell viability was examined directly through a chemiluminescence assay on the chip using the CellTiter-Glo assay. Results: The time needed for the drug screening assay was demonstrated to be less than 30 s for a total of 81 tests. The prediction of optimal drug synergy from the µCA chip was found by matching it to that of the zebrafish MCF-7 tumor xenograft model, instead of the conventional 96-well plate assay. In addition, the critical reagent volume and cell number for each µCA chip test were 200 nL and 100 cells, respectively, which were significantly lower than 100 µL and 4000 cells, which were achieved using the 96-well assay. Conclusion: Our study for the µCA chip platform could improve the high-throughput drug synergy screening targeting the applications of tumor cell biology.