Microarray Microfluidic Platform for

Nov 26, 2018 - Department of Clinical Sciences, College of Veterinary Medicine, Cornell University , Ithaca , New York 14853 , United States. Anal. Ch...
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Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

Nanoliter-Sized Microchamber/Microarray Microfluidic Platform for Antibiotic Susceptibility Testing Morteza Azizi,† Meisam Zaferani,† Belgin Dogan,‡ Shiying Zhang,‡ Kenneth W. Simpson,‡ and Alireza Abbaspourrad*,† †

Department of Food Science, College of Agricultural and Life Sciences, Cornell University, Ithaca, New York 14853, United States Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, United States

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S Supporting Information *

ABSTRACT: The rise of antimicrobial resistance is challenging for physicians in clinical practice to prescribe antibiotics that are effective against bacterial infections. Conventional antibiotic susceptibility testing (AST) is labor-intensive and timeconsuming (18−24 h). Newly emerging technologies such as microfluidics may enable more rapid AST assay time. In this study, we utilize a nanoliter-sized microchamber/ microarray-based microfluidic (N-3M) platform to reduce the AST assay time and rapidly determine the minimum inhibitory concentrations of different antibiotics. Bacterial suspensions, with or without antibiotics, are loaded into small nanoliter-sized chambers, and the change in fluorescent intensity emitted from resazurin reduction, which correlated with bacterial growth, is measured. We demonstrate the reproducibility, functionality, and efficiency of our N-3M platform for numerous wild-type clinical bacterial isolates including Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis. The time-to-result of our N-3M platform varies between ∼1−3 h, depending on growth rates of different bacterial species. We believe that our proposed N-3M platform is robust, is easy-to-implement, has a short time-to-result, and can be applicable for microbial AST in clinical applications.

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channel-embedded cantilevers,26,27 and microchamber/microarray-based microfluidics.28,29 Avesar et al. 28 and Cira et al. 29 have successfully demonstrated microchamber/microarray microfluidic systems for AST assays. However, in their work, the antibiotic solution is loaded into microchambers of the unassembled polydimethylsiloxane (PDMS) microfluidic device by pipetting, followed by freeze-drying overnight, which is a timeconsuming step (t ≥ 24 h). Moreover, the loading process using pipetting is not precise due to the small features of the microchambers. The PDMS microfluidic device is subsequently assembled, adding to the complexity of the process. Moreover, loading the reagents is based on a degassing-driven flow through the microchannel, which is another timeconsuming step (t = 1 h). Due to the small-scale nature of microfluidic systems, these studies have shortened the AST time and improved the assay by increasing the simplicity of the test and decreasing the amount of reagents used; however, a significant period of time is still required for the device and sample preparations. Moreover, the antibiotic type and the antibiotic concentrations must be determined at least 24 h before performing the AST test, which lessens the flexibility of the whole process.

he overconsumption and misuse of antibiotics has been proven to promote bacterial antibiotic resistance, and the emergence of severe, hard-to-treat infections.1−4 More than 2 million people suffer from serious bacterial infections in the United States every year, of which more than 1% (∼20 000) die from resistant bacterial infections. Globally, this issue is projected to cause more than 10 million deaths by 2050 and with huge economic losses2,5 and is considered a global challenge and priority.6 Conventional antibiotic susceptibility testing (AST) approaches, e.g., broth microdilution (BMD)7,8 and Kirby-Bauer disk diffusion,9,10 provide robust AST results but are timeconsuming and labor-intensive.2,11 Consequently, clinical intervention with empirical antimicrobial therapy precedes knowledge of AST, and physicians tend to prescribe antimicrobials with a broad spectrum of activity and the highest possible dosage.12 There is a clear need for novel approaches that would enable rapid, inexpensive, and easily implemented AST without compromising the accuracy. To overcome the limitations of conventional AST, researchers have developed and implemented microfluidic systems that can produce fast results, in a simple and highthroughput fashion that is amenable to point-of-care devices.13−16 A variety of microfluidic platforms have been used for rapid AST, including single-cell imaging,17−21 microwell-based microarrays, 22 droplet-based microfluidics,23,24 asynchronous magnetic bead rotation,25 micro© XXXX American Chemical Society

Received: August 22, 2018 Accepted: November 7, 2018

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Figure 1. Schematic and experimental representations of N-3M platform for AST assay. (a) The schematic structure of the N-3M device. A and B are inlet and outlet, respectively. (b) The schematic AST testing zone is magnified. The I, II, III, IV, V, and VI set is 200 μm, 40 μm, 100 μm, 135°, and 400 μm, respectively. (c) Sequence of bright-field images showing the bacterial suspension loading process. (d) Two consecutive empty microchambers. Microchambers are loaded with (e) bacterial suspension and then (f) antibiotic/resazurin mixture solutions. Flushing the excess bacteria suspension and antibiotic/resazurin from the main channel and isolating microchambers with HFE-7500 in (g) green and (h) red fluorescent modes. Scale bars are 100 μm.

friendly AST system, suitable for quickly deciding to test any antibiotic at any concentration and with a short time-to-result. Therefore, it will have many potential applications in clinical and point-of-care medicine in the near future.

Herein, we propose a novel and simple microfluidic platform based on the microchamber/microarray to test the antimicrobial resistance/susceptibility (R/S) to various antibiotics. Our N-3M platform does not require pretreatment, and sample loading takes ∼3−5 min. The bacterial suspension is loaded by a syringe into side dead-end microchambers using a main channel. Then, it is exposed to a mixture of antibiotic/ resazurin solution. The side microchambers are isolated using a biocompatible oil. Through simple injection with a syringe, all steps of bacterial suspension, antibiotic/resazurin solution, and biocompatible oil loadings are carried out. The readout in our N-3M platform is based on the bacteria cell metabolism and growth, resulting in an increase of fluorescent signal emitted from resazurin reduction (converted to its highly fluorescent counterpart, resorufin). Using our N-3M platform, we perform the AST to find the minimum inhibitory concentrations (MICs) of different antibiotics, including ampicillin, kanamycin, and gentamicin, on four bacterial isolates, clinically isolated from ileal mucosa of patients with Crohn’s disease, including two Escherichia coli (E. coli) (LF82 and T75), one Klebsiella pneumoniae (K. pneumoniae 278), and one Enterococcus faecalis strain (E. faecalis 24). In addition, we develop a robust mathematical algorithm, which quantitatively facilitates the rapid antimicrobial R/S determination. In general, our microfluidic platform is an easily implemented and user-

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EXPERIMENTAL SECTION Materials. SU-8 2015 negative photoresist and developer were obtained from Microchem Corp. (Westborough, MA). Polydimethylsiloxane (PDMS, Sylgard 184) and the PDMS curing agent were purchased from Dow Corning (Midland, MI). Silicon wafers were purchased from University Wafer (Boston, MA). Cellophane tape was purchased from 3 M (Scotch Magic). Polyethylene tubing (i.d. = 0.38 mm, o.d. = 1.09 mm), 27-gauge syringe needles, and 3 mL Luer-Lok tip disposable syringes were purchased from Becton Dickinson (Franklin Lakes N). Resazurin, ampicillin, kanamycin, and gentamicin were purchased from Sigma-Aldrich (St. Louis, MO). Fabrication of PDMS Microfluidic Devices. Following the instruction provided by Microchem (Westborough, MA), the mold was patterned on the silicon wafer using the wellestablished soft lithography technique.14,30,31 In detail, a 40-μm layer of photoresist (SU-8 2025) was created and patterned on a silicon wafer by the spin-coating and UV-photolithography processes, respectively. The patterned silicon wafer was B

DOI: 10.1021/acs.analchem.8b03817 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry postbaked at 95 °C for 5 min and developed using SU-8 developer. Then, it was washed and air-dried by isopropanol and nitrogen, respectively. We made a slight modification in our microfluidic device fabrication which is illustrated in the Supporting Information. Briefly, a thin but fairly rigid PDMS layer is made by pouring a 5:1 ratio of homogenized and degassed PDMS−curing-agent mixture onto the silicon master and spun at 400 rpm for 3 min to have a thin uncured PDMS layer (∼400−600 μm) and followed by a baking step at 70 °C for 10 min. After partial curing of the primary PDMS layer, another homogeneous PDMS−curing-agent mixture (10:1 ratio) is added to the primary PDMS layer to make a thick but soft PDMS layer (4−5 mm) on the top of the primary layer (followed by baking at 65 °C overnight). It is supposed that the first layer can lessen water evaporation from the culture medium, while the second layer helps to better handle the microfluidic device during the AST assay. The tissue culture bores (provided by Harris UniCore, Tedpella) were used to punch the PDMS device for making the inlets and outlets. The PDMS device was bonded to a glass slide (1 mm thickness) using the oxygen-plasma treatment (1 min). The bonded PDMS device was kept in an oven at 65 °C for 12 h for further stabilization. We also soaked the microfluidic devices in water (at 40 °C) for 30 min before doing any AST assay to let water diffuse into the soft PDMS layer for prolonging the water evaporation from the culture medium. Growth Media, Culture Conditions. We used two kinds of media as growth media in this work: Lysogeny broth (LB) and brain heart infusion (BHI) culture media. E. coli T75 and LF82 and K. pneumoniae 278 were cultured by suspending and inoculating a colony of each strain from a fresh streak plate in 3 mL of 2% LB medium and keeping them on a shaker (shaking at 225 rpm) overnight (12 h) at 37 °C. E. faecalis 24 was shook and incubated in BHI culture medium for 12 h at 37 °C in 2% BHI broth media.32−34 The UV−vis spectrophotometer (NanoDrop, Thermo Scientific, Wilmington, DE) was used to read the concentration of bacterial cells in bacterial suspensions. Antibiotic Susceptibility Experiments. We use an imager platform (ZOE Fluorescent Cell Imager, Bio-Rad, Hercules, CA) to read the fluorescent intensity imitated from bacteria culture medium by the occurring redox reaction of resazurin. The powdered stocks of antibiotics were used to prepare antibiotic solutions for the same day use. Broth Microdilution Test. The gold standard broth microdilution test35 was performed by preparing the antibiotic solutions at their final concentrations from a stock solution of each three-examined-antibiotic. A 200 μL volume of each antibiotic solution was pipetted into each microwell of a 96 MicroWell plate (Falcon, BD Biosciences). Then, 10 μL of each bacterial stock suspension was added to each microwell containing antibiotic solution. We incubated the bacteria in the presence of antibiotics for 20 h, and the MIC and susceptibility time was measured upon 80% reduction in OD600-growth curves than the negative control (without adding any antibiotic to bacterial suspension). We performed the broth microdilution test in triplicate.

cm). In both longitudinal sides of the main channel, there are micrometer-sized chambers connected to the main chamber using side microchannels. Each device contains 50 microchambers (25 microchambers on each side); for better visualization, 14 microchambers, 7 microchambers on each side, were schematically shown in Figure 1a. Ports “A” and “B”, as shown in Figure 1a, function as the inlet and outlet, respectively. The dimensions of the bacterial trapping microchambers are shown in more detail in Figure 1b. The diameter and the height of each microchamber are 200 and 40 μm, respectively (shown as “I” and “II” in Figure 1b). The microchambers are connected to the main channel by side microchannels, which have 100 μm × 50 μm dimensions in length (on average) and width, respectively (shown as “III” and “IV” in Figure 1b). The main channel is intersected by the side channel at an angle of 135° (“V” in Figure 1b). The distance between every two consecutive microchambers is 400 μm (“VI” in Figure 1b). As we first inject the bacterial suspension into the main channel, air quickly becomes trapped inside the microchambers, thereby preventing the bacterial suspension itself from being readily loaded into the microchambers. The trapped air has no way to escape except through the walls of the PDMS microchamber itself. Given the fact that cross-linked PDMS has a high fractional free volume (∼34%),36 by applying a slight force during the injection of the bacterial suspension, we can rapidly push the trapped air through the PDMS wall irreversibly (Figure 1c). Figure 1d demonstrates two empty consecutive microchambers before loading the bacterial suspension. After loading the bacterial suspension into the side microchambers (Figure 1e), the main channel is washed out with resazurin/antibiotics solution to (i) unload bacteria from the main channel and (ii) allow the antibiotic/resazurin solution to diffuse into the microchambers (Figure 1f). The resazurin/antibiotic solution diffuses into the side microchambers in a matter of minutes, forming a homogeneous solution of bacteria, resazurin, and antibiotics within the microchambers, at which point the AST assay begins. The main channel is then flushed using a biocompatible oil (HFE-7500) to isolate the microchambers and prevent fluorescence indicator exchange, which would bias the fluorescence intensity readout (green and red fluorescent images representing entrapped bacteria and resazurin/antibiotic solutions in the microchambers are shown in Figure 1g,h, respectively). However, because of the high surface areato-volume ratio of the microchamber and the fractional free volume of PDMS, the rate of water evaporation from the culture medium in the microchambers is high during the AST assay (for instance, ∼50 vol % of the loaded culture medium is evaporated in ∼30 min). Consequently, this evaporation may concentrate the fluorophore and lead to perceived fluorescence increases and biased results (Figure S-1). To fix this problem, we modified the PDMS device assembly by fabricating a duallayer rigid (5:1) and soft (10:1) PDMS device (Figure S-2). After the modification of the fabrication process, we showed that the water evaporation from the microchambers was significantly decreased (∼2% of the loaded culture medium was evaporated after ∼3 h incubation, Figure S-3). Reproducibility of the N-3M Platform. We also demonstrated the reproducibility of the N-3M platform because it is important that the same number of bacteria on average be injected into each microchamber due to a direct correlation between bacterial cell growth and the fluorescence intensity change. Therefore, we injected different concen-

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RESULTS AND DISCUSSION N-3M Design and Loading Criteria. We use the conventional soft-lithography30 technique to fabricate our N3M device, shown schematically in Figure 1a. The N-3M platform consists of a main microchannel (1.21 cm × 0.50 C

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Analytical Chemistry trations (5 × 105, 1 × 106, 5 × 106, 1 × 107 CFU/mL) of green fluorescent protein (GFP)-labeled E. coli LF82 and visually counted the number of bacteria in each microchamber for each concentration (Figure 2a). Since the microchambers are 40 μm

prior to performing the AST assay. Thus, four different concentrations of resazurin in the culture medium were tested, namely, 10 μg/mL (100% X), 1 μg/mL (10% X), 0.4 μg/mL (4% X), and 0.2 μg/mL (2% X) (Figure 3a). Increasing the amount of resazurin from 0.2 μg/mL to 10 μg/mL increased the fluorescence intensity, as expected. In Figure 3a, we drew a yellow dashed line through the microchambers, then measured the fluorescence intensity at each point along the line (n = 10) and converted them into gray values using ImageJ (Figure 3b). Subsequently, the gray values were averaged over the diameter of the microchamber, i.e., the middle 0.6 units of the normalized distance of the yellow dashed line, as shown in Figure 3b, and were correlated with the concentrations of resazurin. The results (Figure 3c) show that the gray values were well-correlated (R2 = 99.93%) with the resazurin concentrations (2% X, 4% X, and 10% X). Based on the correlation trend-line, we found that 25% X or 2.5 μg/mL is the saturation point and there is no fluorescence intensity change at higher resazurin concentrations (Figure S-4). We chose the lowest tested concentration (0.2 μg/mL) of resazurin for further analyses, including device functionality and validation with a range of different bacterial strains. We demonstrated the functionality of our N-3M platform for AST assay of bacterial resistance to the kanamycin antibiotic by loading E. coli LF82 and kanamycin/resazurin solution into the device. Different concentrations of kanamycin were tested on E. coli LF82, including (i) no kanamycin as a negative control (Figure 3d) and with added kanamycin (ii) slightly below the minimum inhibitory concentration (MIC; 10 μg/mL) at 5 μg/ mL (Figure 3e), (iii) slightly higher than the MIC at 20 μg/mL (Figure 3f), and finally (iv) significantly higher than the MIC at 150 μg/mL (as a positive control) (Figure 3g). As expected, the fluorescence intensity increased significantly within 1.5 h incubation time for the negative control in Figure 3d, due to bacterial cell growth in the absence of antibiotic. However, in the presence of kanamycin below the MIC (Figure 3e), bacterial cell growth increased slowly, but the cells eventually survived and thus the fluorescence intensity increased. Using an amount of kanamycin slightly above the MIC (Figure 3f), the fluorescence intensity increased over time (0.5 h) but eventually stopped changing after kanamycin began to affect and prevent further E. coli LF82 bacterial growth (Figure 3f). As shown in Figure 3g, adding kanamycin at a very high concentration above the MIC quickly stopped the bacterial cell growth and thus the fluorescence intensity did not change significantly over the course of the AST assay (Figure 3g). Qualitatively, the experiment successfully confirmed that the N-3M device has good functionality and can be tested for different bacterial strains treated with different antibiotic concentrations. Validation of Quantitative Mathematical R/S Algorithm. Using the N-3M platform, we were able to qualitatively determine the R/S of the E. coli LF82-kanamycin pair and the MIC via the fluorescence intensity change of the culture medium (based on the resazurin reduction) in the microchambers (Figure 3). However, here we propose and demonstrate a quantitative and easy-to-use mathematical algorithm to give us robust antimicrobial R/S results. To use the proposed mathematical algorithm, the fluorescence intensity-based bacterial growth curves obtained by resazurin reduction are needed in both untreated (negative control) and treated bacteria in the presence of antibiotics (positive samples). To validate our algorithm, we used ampicillin-

Figure 2. Reproducibility of N-3M platform for AST assay. (a) Average number of bacteria in each microchamber with different initial concentrations of bacterial suspension into N-3M device. (b) Bacterial cell distribution in each microchamber to show the unbiasedness of microchambers’ position from inlet and outlet on number of bacterial cell in each microchamber.

in height, the loaded bacterial cells can be counted without changing the focal plane of the microscope. Thus, we counted the loaded bacterial cells (10 microchambers in each experiment and 5 experiments for each bacterial loading concentration) at different bacterial cell concentrations using ImageJ and averaged the number of bacterial cells in each microchamber based on the initial bacterial cell concentration. In total, 2 ± 1, 4 ± 2, 14 ± 3, and 25 ± 6 of E. coli LF82 cells were loaded into each microchamber, which correlates with the initial bacterial loading concentrations of 5 × 105, 1 × 106, 5 × 106, and 1 × 107 CFU/mL, respectively. We also loaded GFPlabeled E. coli LF82 (initial concentration of 1 × 107 CFU/ mL) into the device and imaged different microchambers at different positions of the N-3M platform. We demonstrated that the position of the microchamber in the N-3M device (e.g., the first microchamber vs last microchamber) did not affect the number of loaded bacteria within (Figure 2b). The results showed a uniform distribution of E. coli LF82 in each microchamber, with no significant correlation between the microchamber position and bacterial load. Resazurin Concentration Optimization and Qualitative Investigation of the N-3M Functionality. A high initial concentration of resazurin in the microchambers can lead to saturation of the fluorescent signal, making it difficult to discern changes in intensity and compromising the antibiotic screening abilities of the N-3M device. Therefore, it is necessary to find an appropriate initial resazurin concentration D

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Figure 3. Functionality of N-3M platform for AST assay. (a) Loading the resazurin/culture medium mixture into the microchamber at 4 different concentrations (10 μg/mL (100% X), 1 μg/mL (10% X), 0.4 μg/mL (4% X), and 0.2 μg/mL (2% X). (b) Average gray values corresponding to each resazurin/culture medium mixture concentration, quantified by converting fluorescent intensities using ImageJ. (c) Average gray values were averaged on a 0.6 normalized unit of the screened area (plateau regions of the yellow dashed line). Loading microchambers with wild-type E. coli LF82 and resazurin/kanamycin mixture at different concentrations and screening the fluorescent intensity change every 30 min for 1.5 h, including (d) no kanamycin (negative control), (e) 5 μg/mL kanamycin, (f) 20 μg/mL kanamycin, and (g) 150 μg/mL kanamycin. Scale bars are 100 μm.

resistant E. coli T75 and gentamicin-susceptible E. faecalis 24 tested at different concentrations of ampicillin and gentamicin, respectively (Figure 4a,b). Initially, we converted the fluorescence intensities obtained from the culture medium in the microchambers (n = 10 for each treatment) to gray values using ImageJ. We defined a term, δ, by subtracting the average gray value of the positive control from the negative control and dividing the difference by the average gray value of the negative control at the same data collection time-point (eq 1). δ(t = ti) =

GVN(t = ti) − GVP(t = ti) GVN(t = ti)

treatments were quantified for each treatment over time in Figure 4c,d, respectively. We defined two ranges of δ representing antimicrobial R/S: 0 < δ ≤ 0.3 (resistance) and 0.3 < δ < 1.0 (susceptible). To provide deterministic R/S decision-making and shorten the AST assay time, we defined two criteria for resistant bacteria: (i) δ curve should not cross the δ = 0.3 threshold; and (ii) the slope of the δ curve should be approaching zero during the last 0.3 × tAST period, tAST is the AST assay time (e.g., the last 30 min of 100 min of an AST assay). For susceptible bacteria, in addition to being in the 0.3 < δ < 1.0 range, we considered another checkpoint as the consecutive collected δ values should be at least in the 0.3 < δ < 1.0 range for a 0.3 × tAST time-period. Consequently, the susceptibility time is measured as the δ of the lowest tested antibiotic concentration (MIC) crosses the δ = 0.3 value. As shown in Figure 4c, the δ

(1)

in which, GVN and GVP denote the average gray values of the negative and positive samples, respectively, at any data collection time-point (ti). The average δ values corresponding to ampicillin-E. coli T75 and gentamicin-E. faecalis 24 E

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Figure 4. Algorithm for antimicrobial R/S decision making applicable on N-3M platform. (a and b) Average gray values vs incubation time for (a) E. coli T75 under ampicillin and (b) E. faecalis 24 under gentamicin treatments at different concentrations of antibiotics. (c and d) δ curves vs incubation time calculated using our developed algorithm for (c) E. coli T75/ampicillin pairs and (d) E. faecalis 24/gentamicin pairs. (e and f) Cell density (growth curves) based on the optical density (OD600) obtained by the gold standard BMD approach for (e) E. coli T75/ampicillin pairs and (f) E. faecalis 24/gentamicin pairs.

curves of E. coli T75 never crossed the threshold (δ = 0.3) and approach zero during the last 20 min (0.3 × tAST) of the AST assay time (70 min). In contrast, the δ curves of E. faecalis 24, Figure 4d, crossed the targeted threshold (δ = 0.3) at antibiotic concentrations of ≥30 μg/mL (MIC) after 46 ± 3 min (at 30 μg/mL treatment) of incubation time (susceptibility time). Thus, E. coli T75 and E. faecalis 24 were determined as ampicillin-resistant and gentamicin-susceptible, respectively. We used BMD, the gold standard, to verify our developed antimicrobial R/S mathematical algorithm for the N-3M platform. Figure 4e,f shows the growth curves of ampicillintreated E. coli T75 and gentamicin-treated E. faecalis 24,

respectively, treated at the same concentrations of antibiotics as performed in the N-3M platform. The cell density (correlated with bacterial growth) curves were obtained based on the optical density (OD600) of the culture medium. According to the BMD standard protocol provided by the Clinical and Laboratory Standards Institute (CLSI),19 an 80% decrease in cell density (OD600 data) of the treated culture vs the negative control (no antibiotic added) shows bacterial susceptibility to antibiotics. BMD data for the ampicillintreated E. coli T75 and gentamicin-treated E. faecalis 24 in Figure 4e,f show that E. coli T75 is resistant to ampicillin while E. faecalis 24 is susceptible to gentamicin, similar to the results F

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susceptible to kanamycin and gentamicin, while they are resistant to ampicillin (Figure 5a−c). The kanamycin MICs of E. coli T75 and LF82 and K. pneumoniae 278 were the same at 10 μg/mL, while they showed susceptibility to gentamicin at 2, 2, and 5 μg/mL concentrations, respectively (Figure 5a−c). The susceptibility times obtained for E. coli T75 and LF82 and K. pneumoniae 278 under kanamycin treatment were 78 ± 5, 63 ± 6, and 190 ± 9 min. While the susceptibility times of these three strains (E. coli T75 and LF82 and K. pneumoniae 278) under gentamycin treatment were 44 ± 3, 45 ± 6, and 107 ± 8 min, respectively. E. faecalis 24 showed susceptibility to all three ampicillin, kanamycin, and gentamicin treatments. The ampicillin, kanamycin, and gentamicin MICs were determined to be 2, 30, and 30 μg/mL, with susceptibility times of 94 ± 4, 63 ± 5, and 46 ± 3 min, respectively (Figure 5d).

from the N-3M platform (Figure 4c,d). Thanks to the BMD R/S criterion (80% decrease in the cell density, OD600 curve, of the antibiotics-treated bacteria compared to the negative control), the MIC of gentamicin-treated E. faecalis 24 was found to be 30 μg/mL. Consequently, comparison between the MIC and susceptibility time readouts from the N-3M platform (Figure 4c,d) and BMD method (Figure 4e,f) clearly showed a good agreement between our proposed algorithm/ N-3M platform and the gold standard BMD technique. Antimicrobial R/S Determination of Clinically-Isolated Bacteria/Antibiotic Pairs. We performed antimicrobial R/S testing using our verified antimicrobial R/S algorithm/N-3M platform for four bacterial isolates, clinically isolated from ileal mucosa of patients with Crohn’s disease, including E. coli T75 and LF82, K. pneumoniae 278, and E. faecalis 24.37 The antimicrobial R/S determinations of the bacterial strains were conducted using three different antibiotics: ampicillin, kanamycin, and gentamicin. The antibiotics were tested at six different concentrations (2, 5, 10, 30, 70, and 150 μg/mL). Figure 5a−d shows the MICs and the susceptibility times of E. coli T75 and LF82, K. pneumoniae 278, and E. faecalis 24,

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CONCLUSION In conclusion, emerging bacterial resistance to antibiotics has made it imperative that we develop fast, high-throughput, and accurate methods to quickly test bacterial antimicrobial R/S to different antibiotics accessible to clinical microbiology laboratories. In this study, we devised a N-3M platform, an easy-to-implement system, for testing different antibiotics against three clinically important bacterial species, E. coli, K. pneumoniae, and E. faecalis. Using a novel loading of bacterial suspension and antibiotic solutions, the dead-end microchambers, connected to the main channel, were loaded with the bacterial suspension and different antibiotics/resazurin solutions to perform AST. A resazurin indicator was incorporated to show whether there was bacterial cell growth through fluorescence intensity changes. In addition, the biocompatible HFE-7500 oil was used to isolate the microchambers. We tested different antibiotics including ampicillin, kanamycin, and gentamicin and developed a mathematical algorithm to find the MIC and susceptibility time for each bacterial strain/antibiotic pair. Our microfluidic platform worked faster (1−3 h) than the gold standard, BMD (12−18 h) and showed comparable accuracy. In addition, the loading of the bacteria and the antibiotic solutions were carried out using minimal equipment (e.g., a syringe). The N-3M platform is also portable and technically simple and can be used in resource-limited healthcare settings as a clinical diagnostic tool for rapid determination of antimicrobial susceptibility.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b03817. Water evaporation from culture medium in microchambers and diffusion through the PDMS device; details of fabrication and modification of 3M platform; water evaporation from culture medium in microchambers after the modification in device fabrication protocol was highly diluted; and resazurin concentrations in LB culture medium vs average gray value at four different concentrations (PDF)

Figure 5. MIC and susceptibility times of bacterial strains treated with three distinct antibiotics, ampicillin (Amp), kanamycin (Kan), and gentamicin (Gen), as measured with the antimicrobial R/S algorithm/ N-3M platform: (a) E. coli T75, (b) E. coli LF82, (c) K. pneumoniae 278, and (d) E. faecalis 24. The green and pink columns represent the MIC and susceptibility times, respectively, of the susceptible bacteria to any specific antibiotic treatment. Gray columns represent the resistant bacteria. Each treatment was triplicated.

respectively. The green and pink columns in this figure represent the MIC and susceptibility time of susceptible bacteria to each antibiotic drug, respectively. Moreover, gray columns demonstrate bacterial resistance to any tested antibiotics (the MIC and susceptibility times were not defined for the resistant bacterial strains). Our results showed that both E. coli strains (T75 and LF82) and K. pneumoniae 278 are

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. G

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Article

Analytical Chemistry

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ORCID

Alireza Abbaspourrad: 0000-0001-5617-9220 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was performed in part at the Cornell NanoScale Facility, a member of the National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (Grant NNCI-1542081).

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DOI: 10.1021/acs.analchem.8b03817 Anal. Chem. XXXX, XXX, XXX−XXX