Device design and principle of operation. (a) A schematic representation of the ladder microfluidic system including an on-chip water bath. Insets showing the key features of the device: (i) the microchamber triplicates, (ii) the modified serpentine mixer, and (iii) the hydraulic resistor pinch on the side channels. (b) The protocol for platform operation: (i) device is loaded with bacteria suspension (blue), then (ii) loaded with culture medium containing antibiotic (red) and culture medium alone (blue) from two different inlets. The LCGG automatically generates a stable exponential decay concentration gradient of antibiotic, which diffuses into the microchambers. Finally, (iii) the channels are washed with oil (yellow), after a certain loading time, to isolate the microchambers. (c) The fluorescent intensities of resazurin in selected microchambers (M1–M9; PC = positive growth control) changes after 4–5 h of incubation at 37°C indicating bacteria metabolism. Three scenarios are possible where the test shows (i) a minimum inhibitory concentration (MIC) for the bacteria/antibiotic combination, (ii) resistance or MIC higher than the testing range, and (iii) invalid result due to no growth of bacteria. Credit: Communications Engineering (2023). DOI: 10.1038/s44172-023-00064-5
The possibility of rapidly identifying antibiotic resistant bacteria can play a significant role in solving the global antibiotic crisis by facilitating the targeted and timely administration of pharmaceutical drugs. At present, the process of bacterial infection diagnostics take up to three days, which leads to less effective antibiotic treatment.
In a new report now published in Communications Engineering, Ann V. Nguyen and a research team at the Cornell University, New York, reported the development of a microfluidic system with a ladder-shaped design to generate a two-fold serial dilution of antibiotics, suited for national and international testing standards. The design and application process allowed them to scale-down the testing timeframe of antibiotic susceptibility to about 4–5 hours. The research outcomes were consistent with commercially available kits to provide an adaptable and efficient diagnostic tool for antibiotic susceptibility testing.
Antibiotic resistance and antibiotic susceptibility testing
The effects of antibiotic resistance are world-wide, and are credited to antibiotic overuse in human and veterinary medicine, and is associated with defects in animal handling. Researchers have noted the cause to be due to overprescribed antibiotics, and a lack of rapid laboratory tests and the circulation of prescription antibiotics that are only marginally effective. The capacity to develop and use rapid diagnostic tests can identify and characterize resistant bacteria to combat antibiotic resistance. The existing workflow of diagnosing antibacterial infection usually takes up to 2–3 days, during which a patient undergoes isolation, sensitivity, and identification.
While the process begins with sample collection, biochemists can identify the infective agent depending on the characteristics of the pathogen, in a process that takes up to two days in a clinical microbiology lab. Researchers can isolate bacteria to conduct antibiotic susceptibility tests that take up another 16–20 hours resulting in a lack of timely acquisition of the information on antibacterial resistance. 
Computational fluid dynamics simulation and the pressure nodes network. (a) Flow distribution in the ladder. In the inset unique numbers are assigned to the pressure nodes before and after each resistor feature, and at the junctions. The red arrows show the direction of the flow. (i and k) are counters for the resistors and nodes, respectively. (b) Contours of pressure for constriction of (i) L = 420 μm (ii) L = 1650 μm (iii) L = 4950 μm and (iv) the standard mixer resistors for the flow rate of 130 μL h−1 for each of the channels. (c) The pressure at the inlet of the features in A. (d) Hydraulic resistance of the constriction, as a function of the length for width of 40 μm. Simulations for seven constrictions are shown (L = 420 μm, 1649 μm, 4070 μm, 4949 μm, 7990 μm, 10795 μm). At L = 655 μm, the hydraulic resistance of the constriction is equal to that of the mixer. (e) Ratio of the flow rates of the drug and the diluent at their respective inlets will affect the concentration profile of the microchambers. These results are from network simulations. (f) The dilution factor in the Concentration(%)=50×E1−i needs to be E = 2, where i is the chamber number. The formula is fitted to the results in the part E to solve for E using least square algorithm. Credit: Communications Engineering (2023). DOI: 10.1038/s44172-023-00064-5
Microfluidic platforms and the design of a microfluidic ladder-like instrument
Bioengineers have therefore developed microfluidic platforms as a method to improve the sensitivity and speed of testing antibiotic susceptibility. In this work, Nguyen and the team designed and developed a microfluidic system with an optimized ladder-like network to combine and distribute culture media and antibiotics in a two-fold serial dilution. They studied the performance of the platform to conduct antibiotic testing, and developed an adaptable, diagnostic instrument. The setup included a microfluidic platform for antibiotic susceptibility testing, which combined nanoliter microchamber-based methods with a ladder-like concentration gradient generator.
The combination provided them with a standard and tunable antibiotic concentration profile to rapidly identify phenotypic antibacterial susceptibility. The researchers created the platform by incorporating a PDMS layer bonded to conventional glass slides, to allow them to test one antibiotic/bacterial combination per device. Each device served as bioreactors to incubate bacteria with antibiotics during the experiments. 
Characterization of the concentration gradient. (a) images of the microchambers showing the process of molecules diffusing into the microchambers from the main channel at t = 0 s and t = 3 min, and after the main channel is washed with oil. (b) the average concentration of resazurin diffused into the microchambers over time, represented as the area under the curve of the concentration profile at each time point (n = 5). (c) the concentration profile formed in ten microchambers of the ladder microfluidic system after a loading time of three min (n = 3). (d) linear correlation between the concentration profiles from computer simulation, of resazurin, fluorescein, and Calcein formed in the ladder microfluidic system, and theoretical 2-fold dilution concentration profile. (e) Comparison of the concentration profiles of resazurin, fluorescein, and Calcein loaded with their specific loading time and theoretical profile. Dash line with blue zone shows the theoretical concentration profile with 5% error. All data are shown as average ±standard deviation. Credit: Communications Engineering (2023). DOI: 10.1038/s44172-023-00064-5
The microfluidic instrument and its principle-of-action
The device contained three openings, including a drug inlet, a negative inlet, and an outlet to create a ladder-like structure. The team added the antibiotic and culture media to the device from opposing directions to dilute antibiotics as it moved across the system from the drug inlet, through a serpentine surface to dilute the solution and mix it with bacteria at specific flow rates. To identify the specific flow rate, the bioengineers calculated the required resistances and used computational fluid dynamics simulations to generate a greater range of dilutions. They then explored the principle-of-action of the microfluidic system with a pre-existing protocol.
The team used a cation-adjusted Mueller Hinton broth with PrestoBlue cell metabolism indicator in the setup. They then loaded the bacterial suspension into the device with a syringe followed by an antibiotic solution to generate a main channel network. They examined the performance of ladder microfluidic systems to determine the minimal inhibitory concentration of antimicrobial substances on the platform and examined bacterial isolates obtained from animal models. The instrument provided a framework to observe a variety of pathogens including Escherichia coli, and Staphylococcus pseudintermedius. In total, the bioengineers tested 206 bacterial samples on the microfluidic cell-sorting platform. 
a Comparison of match vs. un-match between MICs obtained on-chip using the ladder microfluidic system and the gold standard method conducted at veterinary diagnostic lab. Targeted bacteria are Escherichia coli (EC), Proteus mirabilis (PRM), Enterococcus faecalis (EF), and Staphylococcus pseudintermedius (SP). b Detailed percentage of matched vs. unmatched for each antibiotic/bacteria combination. c Probability of obtaining an accurate MIC for each antibiotic/bacteria combination. Credit: Communications Engineering (2023). DOI: 10.1038/s44172-023-00064-5
Outlook: Antibiotic susceptibility tests with spiked and clinical samples
Using the instruments, the team examined the possibility of bypassing the bacterial isolation step to conduct antibiotic susceptibility testing with urine samples. They accomplished this with urine samples cultured positive for the presence of a single unknown bacteria. The team determined the capacity for minimal inhibitory concentration from the accompanying clinical samples in 4–5 hours.
In this way, Ann V. Nguyen and colleagues designed and optimized a microfluidic system to perform phenotypic antibiotic susceptibility testing to isolate bacteria from culture plates or directly from urine samples. The instrument maintained a ladder-shaped architecture to generate a two-fold concentration gradient, which followed existing methods of standardization alongside clinically relevant antibiotics and drug combinations. The team conducted bacteria loading, antibiotic, and oil loading on the instrument and included a circuit logic for the first time in the study, to generate a microfluidics concentration gradient. The researchers plan to use the method to facilitate rapid antibiotic susceptibility testing to improve patient outcomes and streamline clinical laboratory workflow in a short timeframe.
The team also envisions integrating additional features to the ladder microfluidic instrument, including automatic loading capacity and sample handling within the ladder-chip for real-time image analysis with improved accuracy.
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