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govindhtech · 11 months ago

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AlphaFold2 Rapid Drug Discovery Vision Break Boundaries

Nuclera wants to use Google DeepMind to expedite the drug discovery process &Vertex AI’s AlphaFold2

The rapid protein access benchtop system from Nuclera, a biotechnology company based in the US and the UK, is being combined with AlphaFold2 (ref 1), a ground-breaking protein structure prediction tool from Google DeepMind, to serve the life science community. AlphaFold2 is hosted on Google Cloud’s Vertex AI machine learning platform.

Since proteins make about 95% of therapeutic targets, there is a growing need to generate various variants of active proteins to facilitate drug development. In particular, the discovery of compounds and biological leads requires accurate protein structure prediction.

The structural biology and drug development industries have been enthralled by AlphaFold2, a groundbreaking artificial intelligence tool that DeepMind published in 2021, since it represents a significant advancement in the accuracy of protein structure prediction (ref 2).

Combining the technologies of Google and Nuclera offers drug developers a new integrated approach to enhance protein construct creation and speed up the drug discovery process. Laser-guided protein design will soon be possible with the availability of high grade structures in a matter of minutes or hours. Furthermore, available will be dependable structures for proteins deemed “impossible” to describe experimentally.

Producing meaningful protein

Drug development relies heavily on the accessibility of proteins for lab-based research, which is notoriously expensive and difficult to acquire, placing time and budget constraints on research potential.

With its benchtop eProtein Discovery technology, Nuclera enables life science researchers to extract active proteins from DNA with the goal of improving human health. By combining digital microfluidics with cell-free protein synthesis on Smart Cartridges, Nuclera’s technology enables quick development on protein projects using an automated, high-throughput benchtop protein access system.

How Nuclera’s AlphaFold2 fits in: guided protein design

AlphaFold2 is an artificial intelligence (AI) model created by DeepMind that predicts the 3D structure of a protein based on its 1D amino acid sequence. It has been widely lauded as a breakthrough in biological research and a step forward in the production of vaccines and synthetic materials.

Nuclera’s cloud-based software will use AlphaFold2, which runs on Google Cloud’s Vertex AI, as a key component to enhance the quality and obtainability of proteins. With the use of Nuclera’s cloud software, users may now determine the best protein designs and conditions for protein scaling based on the results of expression and purification screens.

By providing an additional in silico filter during the experiment design phase, AlphaFold2’s integration with the eProtein Discovery Software improves the quality of constructs screened on the system and increases the likelihood of finding a truly optimal target protein on which to develop discovery programs. Moreover, AlphaFold2 will give users of eProtein Discovery profound insights into potential target protein designs, including any effects on folding, structural characteristics, and drug interactions.

Alphafold2 implementation on Vertex AI pipelines

Even though the AlphaFold2 algorithm has enormous potential, it’s vital to remember that it needs an operational model and serving infrastructure.

Predicting the structure of a protein requires a lot of computing power. Scaling up inference workflows can present difficulties, including as managing experiments, maximizing hardware resource consumption, and minimizing inference elapsed time.

In order to enable inference at scale, the Vertex AI solution for AlphaFold 2 prioritizes the following optimizations:

Parallelizing separate phases in the inference pipeline optimizes it.

utilizing the best hardware platform for each stage to maximize hardware usage and, consequently, expenses. The solution automatically provisioned and deprovisioned the computing resources needed for a step as part of this optimization.

presenting a strategy to experiment tracking that is both flexible and resilient, making it easier to execute and analyze hundreds of concurrent inference operations.

In order to create a scalable and resource-efficient AlphaFold pipeline, Nuclera will leverage the Vertex AI platform. Additionally, the company will leverage other Google Cloud services to expose the pipeline via an API and link it with its eProtein Discovery system.

What does the implementation setup entail?

The first goal that AlphaFold2 and Nuclera will accomplish is building a scalable API service that will allow users to access an AlphaFold2 instance running on Google Cloud. Secondly, a dashboard for analytics will be developed that will enable users to compare projected 3D structures for protein variations both statistically and graphically. Third, a feature called “protein of interest” (POI) recommendation will use intelligent selection algorithms to suggest to users potential synthetic protein variants (isoforms, truncations, mutations, orthologs, or fusions), while accounting for a number of constraints like conserved domains or computationally generated scores.

The relevance of eProtein Discovery/AlphaFold2’s application

With the aid of AlphaFold2’s 3D structural insights, Nuclera and its clients will be able to enhance the efficiency of their protein variant manufacturing procedure and learn more about the intricate relationships that exist between residues and the 3D folding protein structure.

The composite predictions provided by the AlphaFold2 module of the eProtein Discovery Software will help customers all over the world gain a better understanding of their proteins and make more informed decisions more quickly. This will ultimately save time needed to advance academic research and successfully find new drugs.

“AlphaFold2 integrated with Nuclera’s eProtein Discovery System is a really exciting demonstration of its practical use in drug discovery, enabling researchers to rapidly and efficiently design and produce proteins with the desired structure and function,” said Shweta Maniar, Global Director, Healthcare & Life Sciences Solutions, Google Cloud.

They are thrilled to be leading the way in the development of AI/ML-assisted drug discovery tools, which believe will accelerate the development of next-generation treatments. They are collaborating with Google Cloud and utilizing the amazing capabilities of AlphaFold2. Visit our GitHub repository to explore this solution in more detail and to test the universal and monomer pipelines that are included. The repository’s artifacts are made to allow for customization.

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#parylene film #parylene coating #parylene conformal coating equipment

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fumpkins · 2 years ago

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New smartphone clip-on can detect Zika virus in blood samples -- LiveScience.Tech

As seen with the COVID-19 pandemic, detection methods that are rapid, simple, accurate, and sensitive are vital for detecting viral pathogens and for controlling the spread of infectious diseases. Unfortunately, laboratory-based methods often require trained personnel and involve complex procedures. In a new study, researchers at the University of Illinois Urbana-Champaign have combined their efforts to develop an instrument that can be clipped on to a smartphone to rapidly test for Zika virus in a single droplet of blood.

Zika virus is primarily transmitted through Aedes aegypti mosquitoes. Although the disease is largely asymptomatic or results in mild symptoms in adults, it causes developmental disorders in newborn babies if their mothers are infected during early pregnancy. Currently, the virus is circulating in more than 87 countries, infecting thousands of people annually, necessitating better testing and control measures.

“Mosquito-borne viruses cause serious diseases, but they have similar symptoms. If you have Zika, malaria, dengue, or chikungunya, you just might show up to the doctor with a fever and they won’t know why,” said Brian Cunningham (CGD Director/MMG), the Intel Alumni Endowed Chair of Electrical and Computer Engineering. “But it’s important to know whether it’s Zika, especially if the patient is a pregnant woman, because the consequences to a developing fetus are really severe.”

Zika virus infections are currently detected through polymerase chain reaction tests performed in a laboratory, which can amplify the genetic material of the virus, allowing scientists to detect it. In the new study, researchers used Loop-Mediated Isothermal Amplification to detect the virus in the blood samples using an approach suitable for point-of-care clinics. While PCR requires 20-40 repeated temperature shifts to amplify the genetic material, LAMP only requires one temperature — 65 °C — making it easier to control. Additionally, PCR tests are very sensitive to the presence of contaminants, especially the other components in a blood sample. As a result, the sample is first purified before it can be used. On the other hand, LAMP does not require any such purification step.

A cartridge, that contains reagents required to detect the virus, is inserted into the instrument to perform the test while the instrument is clipped onto a smartphone. Once the patient adds a drop of blood, one set of chemicals break open the viruses and the blood cells within five minutes. A heater below the cartridge heats it up to 65 °C. A second set of chemicals then amplifies the viral genetic material, and the liquid inside the cartridge fluoresces bright green if the blood sample contains the Zika virus. The entire process takes 25 minutes.

“The other cool aspect is that we’re doing the readout with a smartphone,” Cunningham said. “We’ve designed a clip-on device so that the smartphone’s rear camera is looking at the cartridge while the amplification occurs. When there’s a positive reaction, you see little green blooms of fluorescence that eventually fill up the entire cartridge with green light.”

The researchers are now developing similar devices to simultaneously detect other mosquito-borne viruses and are working on making the devices even smaller. “Although our clip-on detector is pretty small, a lot of the space is taken up by the batteries. In the next version, it will be powered by the phone’s battery,” Cunningham said.

The study “Smartphone clip-on instrument and microfluidic processor for rapid sample-to-answer detection of Zika virus in whole blood using spatial RT-LAMP” was published in the journal Analyst and can be found at 10.1039/d2an00438k.

The work was carried out in collaboration with Rashid Bashir (CGD/M-CELS), an Abel Bliss Professor of Engineering; Enrique Valera, a research assistant professor of bioengineering; Minh Do, a Thomas and Margaret Huang Endowed Professor of Electrical and Computer Engineering; and William King, a professor of electrical and computer engineering. The study was funded by the National Institutes of Health and the National Science Foundation Partnership for Innovation program.

New post published on: https://livescience.tech/2022/07/30/new-smartphone-clip-on-can-detect-zika-virus-in-blood-samples-livescience-tech/

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reallyzealousnight · 3 years ago

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Microfluidic Immunoassay Market - worldwide Forecast to 2025

Microfluidic Immunoassay Market by Product type (Lab-on-chip, percent Analyzers, Reagents and Assay), generation, application (Cardiac, Troponin, BNP) - worldwide Forecast to 2025

• The immunoassay is one of the essential analytical techniques based totally on molecular reputation among antibody and antigen that permits for detection of molecules with excessive specificity and sensitivity. Immunoassays are being extensively used for the quantification of target molecules in lots of programs, which include proteomics, medical diagnostics, pharmaceutical research, and primary organic investigations.

• In recent years, the usage of microfluidic chips for immunoassay has been significantly explored. mixture of microfluidics and immunoassays offers a promising platform for various sensitive and automated point-of-care (p.c) diagnostics.

• Microfluidic systems, additionally termed as lab-on-a-chip and the micro general analysis gadget, have shown promising consequences in immunosensor structures. Microfluidic immunosensors offer huge advantages over conventional immunoassay techniques, which includes a excessive surface-to-quantity ratio for green mass transport in immunoreactions, for that reason permitting for immediate evaluation; a miniaturized micro channel measurement to decrease the intake of samples and reagents; and automatic integration with other features, such as pumps, valves, detectors, and mixers to reap a point-of-care (%) goal.

• Global Microfluidic Immunoassay marketplace is predicted to grow at a CAGR of 12.7% from 2019 to reach $2.01 billion via 2025. Microfluidic immunoassay is swiftly developing enterprise with capability of improving patient health in low-aid settings. % tests are accomplished in vital-care settings, ambulatory centers, physician workplace laboratories, or at patient’s domestic. The % enterprise is growing at a faster pace because of demographic shift and want for excessive throughput prognosis in value effective manner. but, the various present percent gadgets need sample pre-treatment before engaging in the take a look at, produce variable effects, and offer both qualitative, semi-quantitative or negative quantitative check effects. This caused rising recognition on achieving specific and quantitative effects; and miniaturization of p.c devices. microfluidic structures (lab-on-a-chip) have attracted a awesome deal of interest as a miniaturized immunodiagnostic platform. major blessings of LOC include consumption of small volumes of reagents and sample; and transport of precise results with fast turnaround time.

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Key Players

The report includes competitive landscape based on extensive assessment of the key strategic developments adopted by the leading market participants in the industry over the past 4 years (2016-2019). The key players profiled in the global microfluidic immunoassay market are Abaxis, Inc. (a subsidiary of Zoetis Inc.), NanÅmix, Inc., Abbott Laboratories, Koninklijke Philips N.V., Biosurfit SA, Micropoint Bioscience, Inc., Siloam Biosciences, NanoEnTek Inc., OPKO Health, Inc., and Quidel Corporation among others.

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Scope of the Report:

Market by Product Type

Microfluidic Analyzers

Microfluidic Cartridges

POC Analyzers

Reagents and Assays

Market by Technology

Electrochemical LOC POC Technology

Optical POC LOC Technology

Market by Application

Troponin

Brain Natriuretic Peptide (BNP)

Other Cardiac Biomarkers

Market by Geography

Japan

China

India

Rest of Asia-Pacific (RoAPAC)

Key questions answered in the report-

How does the LOC/microfluidics diagnostics increase the efficiency and adoption of quantitative POC immunoassay diagnostics?

How are the leading players utilizing the advantages of LOC/microfluidics diagnostics in their products?

What factors are contributing to the frequent usage of cardiac biomarkers in various end users?

How does the adoption of cardiac biomarker differ from other application?

What are the top competitors in this market and what strategies do they employ to gain shares?

What factors are driving growth and which market segments have the most potential for revenue expansion over the forecast period?

What strategies should new companies look to enter this market use to compete effectively?

What are the major drivers, restraints, challenges, and opportunities, in the global microfluidic immunoassay market?

Who are the major players in various countries and what share of the market do they hold?

What are the geographical trends and high growth regions/countries?

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We are the trusted research partners for leading businesses around the world, providing market intelligence focused towards building revenue transformation strategies. Our research is used by Fortune 500 organizations to attain success by scouting next generation revenue opportunities well ahead of their competition.

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apolloptical · 3 years ago

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. This type of lens does not change over time. Simple static lenses can be fabricated straight onto cartridges. For instance, bent surfaces assembled into microfluidic channels can be used as cylindrical lenses.

On the other hand, periodic arrays of microlens arrays can be fitted and attached over a cartridge's detection area. This allows concurrent detection at various sites. Also, lenses with varying focal lengths can be incorporated on microfluidic cartridges.

#microlens array #polymer injection molding #polymer molding

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biomedgrid · 3 years ago

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Biomed Grid | Automatic Generation of Drug Concentration Gradient by a Modified Ink-jet Printer and its Application in Drug Screening

Abstract

Manual preparation of drug concentration gradient is labor-intensive and limits the efficiency of a drug screening to some extent, especially at large scale. In this study, we reported an automatic method to generate drug concentration gradient in 24-well plate. We loaded drug solution in a modified hot ink-jet printer, designed printing pattern in the WORD software, customized the concentration gradient in the WORD software by RGB coding, and finally printed the drug concentration gradient in the 24-well plate. A drug concentration gradient can be automatically generated within 5s in arbitrary pattern. Based on this flexible technology, we developed an ink-jet printer-based drug screening assay. Concentration gradient of 5-fluorouracil, as well as Mcf-7 cells, were printed in the 24-well plate, and the inhibition rate of Mcf-7 cells was measured by MTT method. This was the first time that hot inkjet printing technology was used in drug screening.

Keywords: Bioprinting; Drug screening; Concentration gradient; Ink-jet printer; RGB

Introduction

Preparation of a concentration gradient of drug candidate is a prerequisite to study cell-drug interaction quantitatively. Currently, this step is manually operated in the laboratory, which is labor intensive and annoying. Automation is desirable in practice. Derringer et al. developed an automatic method to generate concentration gradient [1], and Ye et al. [2] used this method in drug screening [2,3], however, this method was only applicable in microfluidic device, not in 96-well or 24-well plate, thus its applications were limited. Previously, we established drug screening device based on biomedical engineering methods [4]. In this study, we developed a method to generate concentration gradient in plate automatically, using normal commercial ink-jet printer and WORD software. We also used the same printer to print the cell suspension, thus we finally developed a drug screening assay based on hot ink-jet printing. Hot ink-jet printing technique is useful for printing cells in biomedical engineering. It works by the principle in which the heating element turns the “biological inks” into bubbles at bottom of the nozzle instantly and the pressure generated makes a certain amount of inks free from the surface tension there and pushed from the nozzle. As an approach for cell printing, hot ink-jet printer has been used in tissue engineering [5], biological microarray construction [6], stem cell research [7] and gene transcription [8]. However, there are few reports in its application in drug screening at cellular level

In this paper, a hot ink-jet printer was custom-modified as an automatic dispensing tool for the engineering of cell arrays and generation of concentration gradient of drugs in a 24-well plate. By means of this apparatus, cell/drug composite arrays can be automatically generated for drug screening. RGB value was used as the output gauge of drug concentrations for the first time. A Mcf-7 cell/5-fluorouracil composite array was engineered to demonstrate the applicability of the hot ink-jet printer in the drug screening.

Experimental

Materials

Hoechst 33342 fluorescence dye and 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Kubolai inc. Beijing. Propidium iodide was obtained from Jingchun technology inc. Shanghai. 5-fluorouracil was purchased from Sigma- Aldrich, United Kingdom. Phosphorate buffer saline (pH7.4) was prepared and sterilized in lab. DMEM cell culture medium and fetal bovine serum from HyClone, New Zealand, was used in this study. Methanol of HPLC grade from local company was used. Ultrapure water was obtained with Millipore water purification system.

Instruments

Canon PIXMA ip 1980 ink-jet printer with PG830 ink box from Canon, Japan, was modified in this study. Inverted fluorescence microscope IX71 from Olympus, Japan, was used. Agilent 1200 HPLC was from Agilent, USA. Synergy H1 microplate reader was from BioTek, USA (Figure 1). A Canon PIXMA ip 1980 printer and a Canon PG830 ink box for biological printing after modification. The white plate was a 24-well plate used as “paper”.

Modification of the Printer and the Cartridge

A Canon PIXMA ip 1980 hot-injet printer was modified as follows the front cover, paper support, rear tray, paper thickness lever and paper guide were dissembled sequentially while cartridge holder and paper sensor being kept intact. The printer after modification was shown as in Figure 1 (left part). Before use, the body of the printer was sterilized and kept inside an ultraviolet-lighted laminar hood overnight. A Canon PG830 ink box was modified as follows: after the top cover of the ink box was ripped from the main body of the box, the sponge inside the ink box was discarded and the metal filter at the bottom of the box was dismantled with the ink reservoir exposed, followed by a thorough cleanup. The ink box after modification was shown as in Figure 1 (right part). Prior to use, the box was emerged in 75% ethanol and air dried in a laminar airflow, followed by being exposed with ultraviolet light for at least 30 mins.

Figure 1: A Canon PIXMA Ip 1980 Printer and a Canon PG830 Ink Box.

Printing Concentration Gradient of 5-Fluorouracil in the 24-Well Plate

10ml 8mg/ml 5-fluorouracil solution was precisely obtained when 0.08g 5-fluorouracil was dissolved in PBS solution in a 10 ml volumetric flask and filtered through a 0.22 μm diameter membrane to remove possible bacteria. This working solution was loaded in the ink box. The printing pattern was designed in the WORD software with designated RGB values. Clicked the “print” button in the WORD software and the concentration gradient of 5-fluorouracil was automated created in the 24-well plate within 5s. We collected the drug solution printed in each well and analyzed their concentrations with HPLC. In a typical setup, a SB-C18 HPLC column (4.6mm diameter, 250mm long) was used and the sample was detected by ultraviolet absorption with a single wavelength of 265 nm. The temperature of the column was set at 35℃. The volume of each sample was 10 μl and the mobile phase was made of methanol and water (5:95) with a flow rate of 1.0ml/min.

Preparation and Printing of Cell Suspension

Mcf-7 cells of the third generation with a high viability was chosen as the main raw material for preparation of the cell suspension. Prior to being loaded into the ink box, a Mcf-7 cell suspension were prepared by pancreatic digestion, centrifuging, collection, counting and re-suspending. The Mcf-7 cell suspensions were prepared at four different concentrations, 3×106, 6×106, 9×106 and 1.2×107/ml. Following the pattern of a round spot with the diameter of 5 mm, the cell suspension with different densities were printed into a 24-well plate with the modified printer as above. Observation was made with an inverted microscope equipped with a X4 zoom objective.

Counting of Printed Cell Numbers and Viability Analysis

400 μl 10% NCBS culture media was added into the plate loaded with the printed Mcf-7 cells to keep their viability. And 10μl 0.4mg/ml Hoechst 33342 solution was added into the plate at 37℃ for 35mins in the dark, followed by the addition of 10 μl 0.2mg/ ml PI solution for another 10 mins. Then the cell suspension in the plate were collected and centrifuged. The cells from the bottom of centrifuge tube were collected and washed with PBS solution for 3 times. Then the cells washed were re-suspended in 1 ml cell culture medium to form a new cell suspension. 20μl new cell suspension was pipetted out and added dropwise onto a cell counter. Counting of cell number and observation of cells were conducted with an inverted fluorescence microscope with a X 10 zoom objective.

Procedure of Drug Screening

Mcf-7 cells with a high viability was selected for construction of cell/drug arrays. The Mcf-7 cell suspension were prepared by pancreatic digestion, centrifuging, collection, counting, and resuspension with a concentration of 9×106/ml. The Mcf-7 cell suspension was loaded into sterilized a PG830 ink box and printed into 5-mm-diameter round spots in a 24-well plate to form a Mcf-7 cell array. 400μl 10% NCBS culture media was immediately loaded into the plate. After being cultured at 37℃ under 5% CO2 for 24hrs, 8 mg/ml 5-fluorouracil solution were printed into the plate which were already loaded with Mcf-7 cells, with different RGB codes. As a result, cell/drug composite arrays (n=3) were formed. After being at 37℃, 5% CO2 for another 48 hrs, 40μl 5mg/ml MTT solution were loaded into the plate and incubated at 37℃ for 4hrs. Then the supernatant was discarded and 500 μl DMSO was added. The plate was shaken for 15mins to achieve DMSO thoroughly dissolved. Then 200 μl supernatant was collected and transferred into a 96-well plate and the adsorption under 490 nm wavelength was recorded.

Results

Printed Concentration Gradient of 5-Fluorouracil

The printing pattern was designed in WORD software, as shown in Figure 2. Each round spot, corresponding to a well in 24-well plate in location, was 5mm in diameter. These round spots varied in RGB values. We printed drugs following the pattern in Figure 2 and measured the concentration of drug in each well. We plotted the drug concentration in each well, as shown in Figure 3. This technology has five advantages compared to manual method

a) It is automatic;

b) We can obtain arbitrary concentration gradient by customizing the RGB values (Figure 3 A-D);

c) The RGB code value was linearly related to the concentration of printed 5-fluorouracil. This can be expressed as Y = -0.7691X+53.975 with R2 being equal to 0.9878 (Figure 3A);

d) It is fast, and a concentration gradient can be generated within 5s;

e) THE hot inkjet printer was cheaper than a micropipette which is used for manual preparation. (Figure 2)

Figure 2: The Printing Pattern Designed in The WORD Software.

Figure 3: Different Types of Drug Concentration Gradient Printed in the 24-Well Plate.

The printing pattern designed in the WORD software. Each row was designed for generation a specific drug concentration gradient. In the first row, the RGB values from left to right were (20,20,20), (30,30,30), (40,40,40), (50,50,50), (60,60,60), (70,70,70). In the second row, the RGB values from left to right were (70,70,70), (20,20,20), (70,70,70), (70,70,70), (20,20,20), (70,70,70). In the third row, the RGB values from left to right were (20,20,20), (20,20,20), (50,50,50), (50,50,50), (70,70,70), (70,70,70). In the fourth row, the RGB values from left to right were (70,70,70), (70,70,70), (50,50,50), (50,50,50), (20,20,20), (20,20,20). (Figure 3) On the top of each plot was the printing pattern designed in the WORD software, and the number below was RGB value. (Figure 4) (from left to right: 3×106/ml, 6×106 /ml,9×106 /ml), scale bar: 500 μm.

Printing of Mcf-7 Cell Suspension

Figure 4: The Bright Field Images of Printed Round Spots of Mcf-7 Cells with Varied Cell Concentration.

Figure 4 shows the printed pattern of Mcf-7 cells, with different concentration of Mcf-7 suspension loaded in the printer. When the concentrations were 3×106 /ml and 6×106 /ml, the printed round spots were incompletely constructed with sparse distributions of cells. It might be caused by the low concentration of cell suspension. When the concentration was 1.2×107/ml, few inks were injected from the nozzle and the printed round spot was hard to be identified. It might be attributed to the high concentration of the cell suspension which jammed the nozzle. However, when the concentration was 9×106/ml, the printed round spot was full of well-distributed cells. It showed that the cell suspension with a concentration of 9×106/ml is the best-performed one and should be adopted in the experiment.

After counting, the average number of cells in each round spot in the cell array was 3×104. After printing, the viability of the cells was the main gauge which reflects the validity of the cell printing process. Hot ink-jet printing was suspected to cause heat damage or mechanical damage on cells [8]. Hoechst33342 is a membrane permissive dye and emits blue fluorescence under 350 nm excitation after combining with DNA in cells. And PI is only capable of penetrating into dead cells. After PI combines with DNA in cells and emits red fluorescence under 543 nm excitation. Therefore, we can investigate the viability of cells when coupling Hoechst33342 and PI dyes after cell printing. As seen in Figure 5, the number of dead cells which emit red fluorescence is negligible and it supports that the negative effect of our cell printing technique on the viability of printed cells can be ignored (Figure 5).

Figure 5: Microscopic observations of printed cells dyed by Hoechst33342(left) and PI (right), under excitations of 350 nm and 543 nm wavelengths, respectively. Scale bar: 500 μm.

Drug Screening Assay Based on Printing

Table 1: Absorbance by MTT and relative inhibition rate (n = 3).

The concentration of the printed 5-fluorouracil can be pretuned with the RGB code setup from the WORD software. A Mcf-7 cell/5-fluorouracil composite array was formed after incubation for 48 hrs. And the inhibition rate of the experimental group at varied concentrations and the control group were tested with MTT method. And the statistics of their optical absorbance at different RGB values and the relative inhibition rates were listed in Table 1. It can be seen from Table 1 that within the range we investigated, the biological viability of the cells in the composite array increased as the RGB value ramped up. In our experimental setup in which RGB code value was kept between 20 and 70, the concentration of the printed 5-fluorouracil increased as the RGB value dropped. In another word, a low RGB value yielded a cell/drug composite array with a relatively high concentration and dose of 5-fluorouracil. Therefore, as the RGB value was lowered, a stronger interaction between Mcf-7 and 5-fluorouracil was achieved, and a higher inhibition phenomenon occurred. This suggested that the Mcf-7 cell/5-fluorouracil composite array established by our method can be used for the inhibition study of 5-fluorouracil at different concentrations and it possessed the potential of evaluating in vitro cellular viability.

Discussion

Cell printing technique has demonstrated remarkable advantages as following :

a) High precision, cells can be precisely localized and distributed in three dimensions [9];

b) Rapid speed, tissue samples with biological viability can be constructed in a short time;

c) High automation grade, it has the possibility to be industrialized at a large scale [10];

d) Simple to be established and operated, it is straightforward to be modified or upgraded [11].

In our experiment, RGB tuning was used for controlling the output doses of 5-fluorouracil and the hot inkjet technique was adopted to build cell/drug composite arrays. And the viability of the cells in the composite arrays were evaluated. Inspired by the model from Boland ‘s group [11,12], a Canon hot inkjet printer and an ink box were successfully modified into a biological printer. And a Mcf- 7 cell suspension with a concentration of 9×106 cells/ml showed the best printing performance in cell distribution and density on the array. The printed cell array demonstrated patterns with the most complete morphology. By testing the concentration of drug solutions with HPLC, we were able to identify the concentration of 5-fluorouracil on the printed matrix produced from different RGB code value. The results show that high-definition Mcf-7 cell arrays can be constructed with our modified printer and ink box. And moreover, the concentration of 5-fluorouracil arrays can be tuned by changing the RGB color code value. The viability of cells by printing is the major indicator for the feasibility of the printing operation. We used Hoechst33342 and PI to dye the printed cells and applied excitations of 350 nm and 543 nm, respectively. The results showed that hot inkjet printing has little damage on the Mcf-7 cells, and they are in consistency with the literatures [13,14]. Also, we used MTT method to identify the inhibition rate of 5-fluorouracil on Mcf- 7 in the cell/drug array. When RGB code value was 30, the dose of the 5-fluorouracil was 30.09±2.69 μg/ml with a relative inhibition rate of 49.02%. These results supported the feasibility of applying the cell/drug printing method in investigating in vitro cellular viability and it provides new possibility of drug screening based on biological printing.

Conclusion

In this study, we developed an automatic method to generate drug concentration gradient in 24-well plate based on hot inkjet printer. The total time was less than 5s. Arbitrary concentration gradient can be easily generated by RGB coding. The cost of printing equipment was very low. This method is expected to make drug screening experiment more user-friendly.

Read More About this Article: https://biomedgrid.com/fulltext/volume5/automatic-generation-of-drug-concentration-gradient-by-a-modified-ink-jet-printer-and-its-application-in-drug-screening.000876.php

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filtration-products · 6 years ago

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Engineers make clear droplets produce iridescent colors

Engineers at MIT and Penn State University have found that under the right conditions, ordinary clear water droplets on a transparent surface can produce brilliant colors, without the addition of inks or dyes.

In a paper published today in Nature, the team reports that a surface covered in a fine mist of transparent droplets and lit with a single lamp should produce a bright color if each tiny droplet is precisely the same size.

This iridescent effect is due to “structural color,” by which an object generates color simply due to the way light interacts with its geometric structure. The effect may explain certain iridescent phenomena, such as the colorful condensation on a plastic dish or inside a water bottle.

The researchers have developed a model that predicts the color a droplet will produce, given specific structural and optical conditions. The model could be used as a design guide to produce, for example, droplet-based litmus tests, or color-changing powders and inks in makeup products.

“Synthetic dyes used in consumer products to create bright colors might not be as healthy as they should be,” says Mathias Kolle, assistant professor of mechanical engineering at MIT. “As some of these dyes are more strongly regulated, companies are asking, can we use structural colors to replace potentially unhealthy dyes? Thanks to the careful observations by Amy Goodling and Lauren Zarzar at Penn State and to Sara’s modeling, which brought this effect and its physical explanation to light, there might be an answer.”

Sara Nagelberg of MIT, along with lead author Goodling, Zarzar, and others from Penn State, are Kolle’s co-authors on the paper.

Follow the rainbow

Last year, Zarzar and Goodling were studying transparent droplet emulsions made from a mixture of oils of different density. They were observing the droplets’ interactions in a clear Petri dish, when they noticed the drops appeared surprisingly blue. They took a photo and sent it off to Kolle with a question: Why is there color here?

Initially, Kolle thought the color might be due to the effect that causes rainbows, in which sunlight is redirected by rain drops and individual colors are separated into different directions. In physics, Mie scattering theory is used to describe the way spheres such as raindrops scatter a plane of electromagnetic waves, such as incoming sunlight. But the droplets that Zarzar and Goodling observed were not spheres, but rather, hemispheres or domes on a flat surface.

“Initially we followed this rainbow-causing effect,” says Nagelberg, who headed up the modeling effort to try to explain the effect. “But it turned out to be something quite different.”

She noted that the team’s hemispherical droplets broke symmetry, meaning they were not perfect spheres — a seemingly obvious fact but nevertheless an important one, as it meant that light should behave differently in hemispheres versus spheres. Specifically, the concave surface of a hemisphere allows an optical effect that is not possible in perfect spheres: total internal reflection, or TIR.

Total internal reflection is a phenomenon in which light strikes an interface between a high refractive index medium (water, for instance) to a lower refractive index medium (such as air) at a high angle such that 100 percent of that light is reflected. This is the effect that allows optical fibers to carry light for kilometers with low loss. When light enters a single droplet, it is reflected by TIR along its concave interface.

In fact, once light makes its way into a droplet, Nagelberg found that it can take different paths, bouncing two, three, or more times before exiting at another angle. The way light rays add up as they exit determines whether a droplet will produce color or not.

For example, two rays of white light, containing all visible wavelengths of light, entering at the same angle and exiting at the same angle, could take entirely different paths within a droplet. If one ray bounces three times, it has a longer path than a ray that bounces twice, so that it lags behind slightly before exiting the droplet. If this phase lag results in the two rays’ waves being in phase (meaning the waves’ troughs and crests are aligned), the color corresponding to that wavelength will be visible. This interference effect, which ultimately produces color in otherwise clear droplets, is much stronger in small rather than large droplets.

“When there is interference, it’s like kids making waves in a pool,” Kolle says. “If they do whatever they want, there’s no constructive adding up of effort, and just a lot of mess in the pool, or random wave patterns. But if they all push and pull together, you get a big wave. It’s the same here: If you get waves in phase coming out, you get more intensity of color.”

A carpet of color

The color that droplets produce also depends on structural conditions, such as the size and curvature of the droplets, along with the droplet’s refractive indices.

Nagelberg incorporated all these parameters into a mathematical model to predict the colors that droplets would produce under certain structural and optical conditions. Zarzar and Goodling then tested the model’s predictions against actual droplets they produced in the lab.

First, the team optimized their initial experiment, creating droplet emulsions, the sizes of which they could precisely control using a microfluidic device. They produced, as Kolle describes, a “carpet” of droplets of the exact same size, in a clear Petri dish, which they illuminated with a single, fixed white light. They then recorded the droplets with a camera that circled around the dish, and observed that the droplets exhibited brilliant colors that shifted as the camera circled around. This demonstrated how the angle at which light is seen to enter the droplet affects the droplet’s color.

The team also produced droplets of various sizes on a single film and observed that from a single viewing direction, the color would shift redder as the droplet size increased, and then would loop back to blue and cycle through again. This makes sense according to the model, as larger droplets would give light more room to bounce, creating longer paths and larger phase lags.

To demonstrate the importance of curvature in a droplet’s color, the team produced water condensation on a transparent film that was treated with a hydrophobic (water-repelling) solution, with the droplets forming the shape of an elephant. The hydrophobic parts created more concave droplets, whereas the rest of the film created shallower droplets. Light could more easily bounce around in the concave droplets, compared to the shallow droplets. The result was a very colorful elephant pattern against a black background.

In addition to liquid droplets, the researchers 3-D-printed tiny, solid caps and domes from various transparent, polymer-based materials, and observed a similar colorful effect in these solid particles, that could be predicted by the team’s model.

Kolle expects that the model may be used to design droplets and particles for an array of color-changing applications.

“There’s a complex parameter space you can play with,” Kolle says. “You can tailor a droplet’s size, morphology, and observation conditions to create the color you want.”

This research was supported, in part, by the National Science Foundation and the U. S. Army Research Office through the Institute for Soldier Nanotechnologies at MIT.

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scitechman · 5 years ago

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Better Treatment for Diabetic Foot Ulcers

People with type 2 diabetes often suffer from poorly-healing infected wounds on their feet. Using existing methods, however, it takes two days to grow a bacterial culture used to identify the pathogens infecting the wound and their antibiotic resistance – and thus to find an effective antibiotic. With the help of a new rapid test developed by Fraunhofer researchers, it will take just one hour to…

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#Fraunhofer Institute #Microfluidic cartridge #Type 2 diabetes

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reallyzealousnight · 3 years ago

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Microfluidic Immunoassay Market by Product type (Lab-on-chip, percent Analyzers, Reagents and Assay), generation, application (Cardiac, Troponin, BNP) - worldwide Forecast to 2025

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