Brookline, MA, United States
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Boardman A.K.,Center for Manufacturing Innovation | Campbell J.,Center for Manufacturing Innovation | Wirz H.,Center for Manufacturing Innovation | Sharon A.,Center for Manufacturing Innovation | And 3 more authors.
PLoS ONE | Year: 2015

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. Here, we report a novel, scaled-up sample preparation protocol carried out in a new microbial concentration device. 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. 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, we show that our 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. © 2015 Boardman et al.


Boardman A.K.,Center for Manufacturing Innovation | Allison S.,Center for Manufacturing Innovation | Sharon A.,Center for Manufacturing Innovation | Sharon A.,Boston University | And 2 more authors.
Analytical Methods | Year: 2013

To accurately diagnose microbial infections in blood, it is essential to recover as many microorganisms from a sample as possible. Unfortunately, recovery of such microorganisms depends significantly on their adhesion to the surfaces of diagnostic devices. Consequently, we sought to minimize the adhesion of methicillin-sensitive Staphylococcus aureus (MSSA) to the surface of polypropylene- and acrylic-based bacteria concentration devices. These devices were treated with 11 different coatings having various charges and hydrophobicities. Some coatings promoted bacterial adhesion under centrifugation, whereas others were more likely to prevent it. Experiments were run using a simple buffer system and lysed blood, both inoculated with MSSA. Under both conditions, Hydromer's 7-TS-13 and Aqua 65JL were most effective in reducing bacterial adhesion. © 2013 The Royal Society of Chemistry.


Kalashnikov M.,Center for Manufacturing Innovation | Lee J.C.,Harvard University | Campbell J.,Boston University | Sharon A.,Center for Manufacturing Innovation | And 3 more authors.
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2012

The emergence and spread of bacterial resistance to ever increasing classes of antibiotics intensifies 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. In this paper, we present a novel microfluidic platform for antibiotic susceptibility testing based on stress-activation of biosynthetic pathways that are the primary targets of antibiotics. We chose Staphylococcus aureus (S. aureus) as a model system due to its clinical importance, and we selected bacterial cell wall biosynthesis as the primary target of both stress and antibiotic. Enzymatic and mechanical stresses were used to damage the bacterial cell wall, and a β-lactam antibiotic interfered with the repair process, resulting in rapid cell death of strains that harbor no resistance mechanism. In contrast, resistant bacteria remained viable under the assay conditions. 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. 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 (Sanger 476) and resistant (MW2) 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. We demonstrate the successful development of a rapid microfluidic-based and stress-activated antibiotic susceptibility test by correctly designating the phenotypes of 16 additional 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. © 2012 The Royal Society of Chemistry.


Campbell J.,Center for Manufacturing Innovation | McGuinness I.,Center for Manufacturing Innovation | Wirz H.,Center for Manufacturing Innovation | Sharon A.,Center for Manufacturing Innovation | And 3 more authors.
Journal of Nanotechnology in Engineering and Medicine | Year: 2015

We have developed a three-dimensional (3D) bioprinting system capable of multimaterial and multiscale deposition to enable the next generation of "bottom-up" tissue engineering. This area of research resides at the interface of engineering and life sciences. As such, it entails the design and implementation of diverse elements: a novel hydrogelbased bioink, a 3D bioprinter, automation software, and mammalian cell culture. Our bioprinter has three components uniquely combined into a comprehensive tool: syringe pumps connected to a selector valve that allow precise application of up to five different materials with varying viscosities and chemistries, a high velocity/high-precision x-y-z stage to accommodate the most rapid speeds allowable by the printed materials, and temperature control of the bioink reservoirs, lines, and printing environment. Our customdesigned bioprinter is able to print multiple materials (or multiple cell types in the same material) concurrently with various feature sizes (100 μm-1 mm wide; 100 μm-1 cm high). One of these materials is a biocompatible, printable bioink that has been used to test for cell survival within the hydrogel following printing. Hand-printed (HP) controls show that our bioprinter does not adversely affect the viability of the printed cells. Here, we report the design and build of the 3D bioprinter, the optimization of the bioink, and the stability and viability of our printed constructs. © 2015 by ASME.


Chatterjee A.,Boston University | Chatterjee A.,Center for Manufacturing Innovation | Mirer P.L.,Center for Manufacturing Innovation | Zaldivar Santamaria E.,Center for Manufacturing Innovation | And 4 more authors.
Analytical Chemistry | Year: 2010

The life science and healthcare communities have been redefining the importance of ribonucleic acid (RNA) through the study of small molecule RNA (in RNAi/siRNA technologies), micro RNA (in cancer research and stem cell research), and mRNA (gene expression analysis for biologic drug targets). Research in this field increasingly requires efficient and high-throughput isolation techniques for RNA. Currently, several commercial kits are available for isolating RNA from cells. Although the quality and quantity of RNA yielded from these kits is sufficiently good for many purposes, limitations exist in terms of extraction efficiency from small cell populations and the ability to automate the extraction process. Traditionally, automating a process decreases the cost and personnel time while simultaneously increasing the throughput and reproducibility. As the RNA field matures, new methods for automating its extraction, especially from low cell numbers and in high throughput, are needed to achieve these improvements. The technology presented in this article is a step toward this goal. The method is based on a solid-phase extraction technology using a porous polymer monolith (PPM). A novel cell lysis approach and a larger binding surface throughout the PPM extraction column ensure a high yield from small starting samples, increasing sensitivity and reducing indirect costs in cell culture and sample storage. The method ensures a fast and simple procedure for RNA isolation from eukaryotic cells, with a high yield both in terms of quality and quantity. The technique is amenable to automation and streamlined workflow integration, with possible miniaturization of the sample handling process making it suitable for high-throughput applications. © 2010 American Chemical Society.


Campbell J.,Center for Manufacturing Innovation | Pollock N.R.,Beth Israel Deaconess Medical Center | Sharon A.,Center for Manufacturing Innovation | Sharon A.,Boston University | And 2 more authors.
Analytical Methods | Year: 2015

We present a lab-on-a-chip and associated instrument for heterogeneous enzyme-linked immunosorbent assay (ELISA)-based detection of proteins from liquid samples. The system performs all necessary ELISA steps (starting from antigen incubation) in a quarter of the time required for corresponding plate-based protocols. We have previously described the instrument, which automates fluidic control via remote valve switching and detects fluorescence from reacted substrate, for use in a molecular diagnostics application. The ELISA chip reported here 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." We show that our 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. © 2015 The Royal Society of Chemistry.


PubMed | Center for Manufacturing Innovation
Type: Journal Article | Journal: Biofabrication | Year: 2017

Due to its relatively low level of antigenicity and high durability, titanium has successfully been used as the major material for biological implants. However, because the typical interface between titanium and tissue precludes adequate transmission of load into the surrounding bone, over time, load-bearing implants tend to loosen and revision surgeries are required. Osseointegration of titanium implants requires presentation of both biological and mechanical cues that promote attachment of and trigger mineral deposition by osteoblasts. While many factors contribute to differentiation, the relative importance of the various cues is unclear. To substantially improve osseointegration of titanium implants, we generated a gelatin methacryloyl (GelMA) scaffold, using an extrusion-based 3D bioprinter, which can be directly printed on and grafted to the titanium implant surface. We demonstrate that this scaffold is able to trigger mineral deposition of both MG63 osteoblasts and primary normal human osteoblasts in the absence of any exogenous osteogenic factors. Films of the same formulation failed to promote mineral deposition suggesting that the three dimensional scaffold was able to tip the balance in favor of differentiation despite other potentially unfavorable differentiation cues of the material. We further show that these GelMA lattices can be directly grafted to titanium alloy and are secure in vitro over a period of seven weeks. When grafted within a groove system, the GelMA hydrogel is protected from shearing forces in a marrow implantation model. This prepares the way for osteogenic coatings to be directly manufactured on the implant surface and packaged for surgery.


PubMed | Boston University, Beth Israel Deaconess Medical Center and Center for Manufacturing Innovation
Type: Journal Article | Journal: Analytical methods : advancing methods and applications | Year: 2015

We present a lab-on-a-chip and associated instrument for heterogeneous enzyme-linked immunosorbent assay (ELISA)-based detection of proteins from liquid samples. The system performs all necessary ELISA steps (starting from antigen incubation) in a quarter of the time required for corresponding plate-based protocols. We have previously described the instrument, which automates fluidic control via remote valve switching and detects fluorescence from reacted substrate, for use in a molecular diagnostics application. The ELISA chip reported here 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. We show that our 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.


PubMed | Center for Manufacturing Innovation
Type: Journal Article | Journal: Lab on a chip | Year: 2012

The emergence and spread of bacterial resistance to ever increasing classes of antibiotics intensifies 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. In this paper, we present a novel microfluidic platform for antibiotic susceptibility testing based on stress-activation of biosynthetic pathways that are the primary targets of antibiotics. We chose Staphylococcus aureus (S. aureus) as a model system due to its clinical importance, and we selected bacterial cell wall biosynthesis as the primary target of both stress and antibiotic. Enzymatic and mechanical stresses were used to damage the bacterial cell wall, and a -lactam antibiotic interfered with the repair process, resulting in rapid cell death of strains that harbor no resistance mechanism. In contrast, resistant bacteria remained viable under the assay conditions. 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. 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 (Sanger 476) and resistant (MW2) 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. We demonstrate the successful development of a rapid microfluidic-based and stress-activated antibiotic susceptibility test by correctly designating the phenotypes of 16 additional 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.


PubMed | University of Massachusetts Boston and Center for Manufacturing Innovation
Type: Journal Article | Journal: PloS one | Year: 2015

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. Here, we report a novel, scaled-up sample preparation protocol carried out in a new microbial concentration device. The process can efficiently lyse 10 mL of bacteremic blood while maintaining the microorganisms viability, giving a 30-L 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. 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, we show that our 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.

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