Microfluidics, Biosensors, Assay Development

In Vitro Diagnostics Projects

Integrated, biological diagnostics on a molecular level

Fraunhofer CMI and their research collaborators from Boston University developed a fully integrated lab-on-a-chip and associated instrument for the detection of bacteria from liquid samples in this prototyping project in biomedical engineering.

Integrated, biological diagnostics on a molecular level
© Photo Fraunhofer CMI

Integrated, biological diagnostics on a molecular level.

Objective

The major challenges of point-of-care clinical diagnostics are sample preparation and detection accuracy. The field of clinical diagnostics is moving toward molecular testing, such as polymerase chain reaction (PCR), which is the most sensitive and specific methodology currently available. On the other hand, PCR suffers from labor intensive sample preparation to isolate nucleic acids and physical separation requirements in a laboratory to minimize sample contamination. Existing point-of-care diagnostics use antibody-based immuno-detection, which enables simple test protocols but lacks the sensitivity and specificity of molecular techniques. To address this challenge, Fraunhofer CMI aimed to design an integrated lab-on-a-chip consumable and a prototype instrument. 

Methodology

The system conducts bacterial lysis, nucleic acid isolation and concentration, polymerase chain reaction (PCR), and end-point fluorescent detection. Approximately the size of a credit card, the cost of the plastic, microfluidic chip is minimized by keeping all active components in the instrumentation (valves, heating units, optics, etc.) and by having a simple, planar layout. A novel porous polymer monolith (PPM) embedded with silica is incorporated onto the chip to lyse bacteria and isolate nucleic acids from clinical samples. The prototype instrument automates fluidic handling, thermal control, and optical detection using a unique, valveless switching fluidic control system.

Results

Integrated chip design includes bacterial and mammalian cell lysis, mixing reagents, isolation and concentration of nucleic acids, PCR, and fluorescence detection

Automation of fluidics, thermal cycling, and optical detection

Low-cost manufacturing design 

Bacterial and viral detections in a point-of-care setting were equivalent in sensitivity and specificity to a hospital's clinical laboratory. 

Collaborators 

Department of Biomedical Engineering, Boston University, Boston, MA 

Funding

This project was funded by the BU-Fraunhofer Alliance for Medical Devices, Instrumentation, and Diagnostics.

A microfluidic platform for rapid, stress-induced antibiotic susceptibility testing

Fraunhofer CMI and their academic collaborators designed, machined, and automated an experimental system for in vitro diagnostics.

Top view of microfluidic chip.
© Photo Fraunhofer CMI

Top view of microfluidic chip.

Side view of microfluidic chip.
© Photo Fraunhofer CMI

Side view of microfluidic chip.

Microscopic imaging of antibiotic susceptibility test.
© Photo Fraunhofer CMI

Microscopic imaging of antibiotic susceptibility test.

Objective

Researchers at Fraunhofer CMI sought to drastically reduce test times by developing a novel and rapid antibiotic susceptibility testing platform.

Need

The emergence and spread of bacterial resistance to ever increasing classes of antibiotics has intensified the need for fast phenotype-based clinical tests for determining antibiotic susceptibility. Standard susceptibility testing relies on the passive observation of bacterial growth inhibition in the presence of antibiotics, and therefore requires long wait times for results (8–24 hours). 

Methodology

Fraunhofer CMI and their collaborators have developed a novel microfluidic platform for antibiotic susceptibility testing that potentiates the action of antibiotics by applying both mechanical and enzymatic stress to the bacteria. 

The scientists chose Staphylococcus aureus (S. aureus) as a model system due to its clinical importance, and selected bacterial cell wall biosynthesis as the primary target of both stress and antibiotic. Bacteria, covalently-bound to the bottom of the microfluidic channel, were subjected to mechanical shear stress created by flowing culture media through the microfluidic channel and to enzymatic stress with sub-inhibitory concentrations of the bactericidal agent lysostaphin. Strains that harbor no beta-lactam resistance mechanism were rapidly killed under these stressful conditions in the presence of oxacillin. In contrast, resistant bacteria remained viable throughout the assay. Bacterial cell death was monitored via fluorescence using the Sytox Green dead cell stain, and rates of killing were measured for the bacterial samples in the presence and absence of oxacillin. Using model susceptible and resistant S. aureus strains, a metric was established to separate susceptible and resistant staphylococci based on normalized fluorescence values after 60 min of exposure to stress and antibiotic. Because this ground-breaking approach is not based on standard methodology, it circumvents the need for minimum inhibitory concentration (MIC) measurements and long wait times. 

Results

The researchers at Fraunhofer CMI demonstrated the successful development of a rapid microfluidic-based and stress-activated antibiotic susceptibility test by correctly designating the phenotypes of 18 clinically relevant S. aureus strains in a blinded study. In addition to future clinical utility, this method has great potential for studying the effects of various stresses on bacteria and their antibiotic susceptibility.

Academic Collaborators

Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA

Department of Mechanical Engineering, Boston University, Boston, MA

Department of Biomedical Engineering, Boston University, Boston, MA

Supporters/Funding

The project described was supported in part by Award Number R21AI079474 from the National Institute of Allergy and Infectious Diseases (NIAID). Expansion of this work is currently being funded by Award number R01AI101446 from the NIAID.  The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. The project was also supported by Fraunhofer USA.

Publications

Kalashnikov, M., J.C. Lee, J. Campbell, A. Sharon, and A.F. Sauer-Budge. A microfluidic platform for rapid, stress-induced antibiotic susceptibility testing of Staphylococcus aureus. Lab Chip, 2012. 12(21): 4523-32. PubMed PMID: 22968495.

Kalashnikov M, Campbell J, Lee JC, Sharon A, Sauer-Budge AF. Stress-induced Antibiotic Susceptibility Testing on a Chip. J Vis Exp. 2014(83). PubMed PMID: 24430495.

Future Directions

The platform is currently being expanded to include other bacteria and antibiotics.

Development of an automated on-chip bead-based ELISA platform

In this In Vitro Diagnostics project, Fraunhofer CMI and their research collaborators worked to design and automate a prototype of a microfluidic ELISA lab-on-a-chip device.

Chip to instrument interface.
© Photo Fraunhofer CMI

Chip to instrument interface.

Cartoon of assay design.
© Photo Fraunhofer CMI

Cartoon of assay design.

Objective

The rapid detection of protein analytes from patient samples is critical for proper diagnosis of numerous diseases. Fraunhofer CMI and their collaborators sought to develop a lab-on-a-chip assay and associated instrument for the heterogeneous enzyme-linked immunosorbent assay (ELISA)-based detection of proteins from liquid samples. 

Method

The system performs all necessary ELISA steps (starting from antigen incubation) in a quarter of the time required for corresponding plate-based protocols. The instrument, automates fluidic control via remote valve switching and detects fluorescence from reacted substrate, for use in a molecular diagnostics application. The ELISA chip utilizes a high surface area bead bed to enhance capture efficiency and increase the dynamic range of the assay as compared to a standard plate-based ELISA. Its functionality is demonstrated using human IL-10 as a model antigen, but theoretically any sandwich ELISA could be ported onto this “open source platform.” 

Results

Fraunhofer CMI’s scientists show that their automated on-chip assays have greater sensitivities than the corresponding standard manual plate-based ELISAs, and that single samples can be assayed in a fraction of the time.

Academic Collaborators

Nira Pollock, MD PhD.  Boston Children's Hospital/Beth Israel Deaconess Medical Center, Boston, MA, USA

Department of Mechanical Engineering, Boston University, Boston, MA, USA

Funding

This work was supported, in part, by NIH/NIAID grant 1 K23 AI074638-01A2. Boston University and Fraunhofer Gesellschaft also funded this project in part through the BU-Fraunhofer Alliance for Medical Devices, Instrumentation, and Diagnostics Program. Additional funds for early project work came from the Pittsfeld Anti-Tuberculosis Association and Harvard Catalyst.

Publication

Campbell J, Pollock NR, Sharon A, Sauer-Budge AF. Development of an automated on-chip bead-based ELISA platform. Anal. Methods 2015; 7:8472–7. DOI: 10.1039/C5AY00264H.

Rapid Microbial Sample Preparation from Blood Using a Novel Concentration Device

Fraunhofer CMI and their collaborators have designed, fabricated, and tested a new microbial concentration device for in vitro diagnostics.

Purification and concentration of bacteria from blood
© Photo Fraunhofer CMI

Purification and concentration of bacteria from blood

Automated instrument design.
© Photo Fraunhofer CMI

Automated instrument design.

Objective

Researchers at Fraunhofer CMI sought to develop a device that could rapidly isolate and concentrate viable microorganisms from infected blood as a universal sample preparation device for downstream microbial diagnostics..

Need

Appropriate care for bacteremic patients is dictated by the amount of time needed for an accurate diagnosis. However, the concentration of microbes in the blood is extremely low in these patients (1–100 CFU/mL), traditionally requiring growth (blood culture) or amplification (e.g., PCR) for detection. Current culture-based methods can take a minimum of two days, while faster methods like PCR require a sample free of inhibitors (i.e., blood components). Though commercial kits exist for the removal of blood from these samples, they typically capture only DNA, thereby necessitating the use of blood culture for antimicrobial testing. 

Methodology

Fraunhofer CMI present a novel, scaled-up sample preparation protocol carried out in a new microbial concentration device. It is a that utilizes lysis solutions and centrifugation to selectively remove the blood components while maintaining the viability of the microorganisms. After each spin, the pellet (containing concentrated microorganisms) is protected in the unique T-shaped slider valve during the removal of the supernatant. The valve is then actuated, revealing the pellet. The process can efficiently lyse 10 mL of bacteremic blood while maintaining the microorganisms’ viability, giving a 30‑μL final output volume. A suite of six microorganisms (Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, and Candida albicans) at a range of clinically relevant concentrations was tested. 

Results

All of the microorganisms had recoveries greater than 55% at the highest tested concentration of 100 CFU/mL, with three of them having over 70% recovery. At the lowest tested concentration of 3 CFU/mL, two microorganisms had recoveries of ca. 40–50% while the other four gave recoveries greater than 70%. Using a Taqman assay for methicillin-sensitive S. aureus (MSSA) to prove the feasibility of downstream analysis, they show that the microbial pellets are clean enough for PCR amplification. PCR testing of 56 spiked-positive and negative samples gave a specificity of 0.97 and a sensitivity of 0.96, showing that our sample preparation protocol holds great promise for the rapid diagnosis of bacteremia directly from a primary sample. Additional work has shown that isolated bacteria can be accurately identified using Surface-enhanced Raman Spectroscopy and our microfluidic antibiotic susceptibility test.

Academic Collaborators

Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA

Department of Chemistry, Boston University, Boston, MA

Department of Mechanical Engineering, Boston University, Boston, MA

Department of Biomedical Engineering, Boston University, Boston, MA

Funding

This project was supported in part by Award Number R01AI090815 from the National Institute of Allergy and Infectious Diseases (NIAID). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. The project was also supported by Fraunhofer USA.

Publications

Boardman AK, Campbell J, Wirz H, Sharon A, Sauer-Budge AF. Rapid Microbial Sample Preparation from Blood Using a Novel Concentration Device. PLoS ONE. 2015;In Press.10:e0116837. PubMed PMID: 25675242; PubMed Central PMCID: PMC4326418.

Sauer-Budge, A.F., A.K. Boardman, S. Allison, H. Wirz, D. Foss, and A. Sharon. Materials and surface properties optimization to prevent biofouling of a novel bacterial concentrator. Procedia CIRP. 2013;5:185-8.

Premasiri, W.R., A.F. Sauer-Budge, J.C. Lee, C.M. Klapperich, and L.D. Ziegler. Rapid bacterial diagnostics via surface-enhanced Raman spectroscopy. Spectroscopy Solutions for Materials Analysis, 2012. June: 8-21.

Academic Collaborators

Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA

Department of Chemistry, Boston University, Boston, MA

Department of Mechanical Engineering, Boston University, Boston, MA

Department of Biomedical Engineering, Boston University, Boston, MA

 

Funding

This project was supported in part by Award Number R01AI090815 from the National Institute of Allergy and Infectious Diseases (NIAID). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. The project was also supported by Fraunhofer USA.

 

Low-cost, real-time, continuous flow PCR system for pathogen detection

Fraunhofer CMI (USA) and their partners from Fraunhofer IPT (Germany), as part of the ML2 consortium, aim to create portable, low-cost pathogen diagnostics by combining microfluidic and electronic layers into a single device.

© Photo Fraunhofer CMI

Photograph of the microfluidic layer showing the 31-mm x 46-mm footprint. The total volume of the microfluidic channel is 20 µL.

© Photo Fraunhofer IPT

Photograph of the completed device with gold-printed heaters, microfluidic channels, and fluidics connector.

© Photo Fraunhofer IPT

Photograph of the drum barrel used to hot emboss microfluidic features during roll-to-roll production.

Objective

Bacterial resistance to antibiotics is escalating and represents a significant health threat to the human population. These alarming circumstances have largely resulted from the current clinical practice of prescribing broad-spectrum antibiotics first and identifying whether or not they are needed or even useful later—after the standard 18-48h culture. When it is critical to know whether or not a particular pathogen is causing disease, as is the case for outbreaks or epidemics, clinicians and laboratories turn to a rapid nucleic acid test (NAT) that can identify a pathogen within hours. However, NATs require specialized lab space, expensive reagents, and extensively trained technicians to appropriately conduct the assays. To reduce costs, labs process samples in batches once-per-day making the effective turn-around-time 24h, by which time patients have left the clinic, often with broad-spectrum antibiotics in hand. In order to stem the tide of antibiotic resistance and preserve this critical resource for the correct patient, rapid point-of-care diagnostics are essential. To address this need, Fraunhofer CMI (in conjunction with Fraunhofer IPT) has developed a microfluidic chip for NATs that can identify pathogens within 20 minutes and can be produced at low-cost, thus eliminating batch processing altogether. 

Methodology

The pathogen sample is introduced into a small 3 cm x 5 cm thermoplastic chip and mixed with NAT reagents in a microfluidic channel. The genetic content of the sample is amplified as it moves through the microfluidic channel, which traverses two heating zones. Within 20 minutes, the presence of the pathogen is detected on-chip by fluorescence. The final device incorporates both heating elements and microfluidics into separate layers that can be laminated en masse by ML2 industrial-scale roll-to-roll manufacturing methods.

Results

Integrated chip design consists of both microfluidic and heating layers for portably, point-of-care pathogen detection

Fluorescence is monitored in real-time for the quantitative detection of pathogens at concentrations as low as 10 DNA copies/μL

Method has been validated with two bacterial targets: Chlamydia trachomatis and Escherichia coli O157:H7

Low-cost manufacturing design compatible with roll-to-roll embossing for large scale production

Collaborators

Fraunhofer IPT, Aachen, Germany

Funding

7th EU Framework Programme (Contract No. 318088; ML2: MultiLayer MicroLab)

Publication

Fernández-Carballo, L. B.; McGuiness, I.; McBeth, C.; Kalashnikov, M.; Borrós, S.; Sharon, A.; Sauer-Budge, A.F. "Low-cost, real-time, continuous flow PCR system for pathogen detection", Biomed Microdevices (2016) 18:34; DOI 10.1007/s10544-016-0060-4.

In the News

US Fraunhofer Center Develops Method for Mass Manufacture of Low-Cost Continuous Flow PCR Devices, Mar 30, 2016, Madeleine Johnson for Genome.