Patent Publication Number: US-2023137251-A1

Title: Inductively coupled capacitor wireless sensor system for rapid antimicrobial susceptibility testing

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application claims priority to U.S. Provisional Application No. 63/274,663, filed on Nov. 2, 2021. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention related to biological sensors and, more specifically, to a wireless sensor for detecting the susceptibility of microorganisms to antibiotics. 
     2. Description of the Related Art 
     Drug resistant infections present great challenges, especially in hospital settings where 30% of ICU patients are affected in high-income countries alone and two to three folds higher in low- and mid-income countries. According to a recent WHO report, 2.8 million AMR infections occur each year in the U.S. alone; and at least 1.27 million people died from AMR infection in 2019 worldwide. It is predicted that by 2050, there could be 10 million deaths a year globally if no effective treatment of AMR is available. Rapid detection of AMR has been proven critical for saving sepsis patients. In an animal study that mimics sepsis, it was revealed that antibiotic treatment administered 12 hours after bacterial inoculation resulted in a 96-hour survival rate of 80%, whereas treatment administered at 15 hours had the survival rate down to only 13.3%. 
     Current methods of pathogen detection by blood culture have a median growth time of around 13 hours. Without rapid detection of antimicrobial susceptibility, the patients may be given rather ineffective treatment. This can cause the precious window to prevent patient mortality to be missed due to AMR. Traditional phenotypic method such as dilution methods, agar disk diffusion testing, and gradient diffusion methods, typically takes 1-2 days to generate reliable results. Genotypic ASTs on the other hand, directly detect biomarkers associated with resistance with molecular detection tools including qPCR, whole-genome sequencing, and MALDI-TOF. Such tools are highly sensitive and could produce a report in hours. How-ever, these genomic approaches require detailed knowledge of AMR gene sequences and data analysis in advance, and thus cannot detect newly developed resistance mechanisms  6 . Other new approaches are being developed including optical imaging to identify microbial in microfluidic device, pH sensor for tracking byproducts of microbial growth, bioluminescent assay of ATP, magnetic sensor with antibody coating, and electrochemical biosensors with peptide or anti-body coating. However, these methods require extensive image/data processing, complex sample preparation, and/or large equipment that is difficult to operate in clinical settings. Rapid high-throughput phenotypic ASTs are urgently needed to reduce sepsis mortality and help with the antibiotic stewardship programs. 
     Application of the LC sensor in medical devices dates back to 1967. The magnetic coupling between sensor coil and the detection coil transmits AC electricity to the sensor circuit where the resonant frequency of the inductor (L) and capacitor (C) could be interpreted by analyzing the resonant frequency of the system on a frequency spectrum. The resonant frequency changes in response to the surrounding environment, allowing the detection of substances that change the surface property of the sensor. The wireless nature of the sensor enables sensing in hard-to-access locations either in an instrument or the human body such as a wound health sensor. In addition, LC sensor requires no integrated power to operate. Thus, it is possible to make small form factor LC sensors with long life span. These advantages make LC sensors attractive in designing biomedical devices where the sensors are often sealed and require low to no maintenance to operate in biological environments. However, this technology did not come onto the main stage of remote query systems until the development of Micro Electro-Mechanical System (MEMS). 
     BRIEF SUMMARY OF THE INVENTION 
     The present invention provides a rapid phenotypic AST system based on a wirelessly magnetically coupled LC sensor that can exploit the capacitive nature of bacteria. The present invention thus bypasses the need for targeted sample enrichment surface modifications or coatings, in turn achieving bacterial growth monitoring or killing/inhibition behavioral study to aid in rapid diagnosis of bacterial infection and ASTs. A bacterial culture may be diluted and aliquoted into a well plate having a sensor according to the present invention. A receiver coil connected to an impedance analyzer on the bottom of the well plate wirelessly communicates with the sensor and scans a spectrum of electrical wavelength to find the resonance frequency between the sensor and the receiver coil. The resonance frequency of the system is recorded periodically and combined with the parameter of the sensor to calculate the permittivity of the bacteria culture. The permittivity readout is plotted as a time series, and the slope of the curve over an initial time period is used to access the sensitivity score of bacteria against specific antibiotics. The baseline of the sensitivity score is determined by acquiring the growth slope of sterile LB media with treatment and known sensitive strain under treatment. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) 
       The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which: 
         FIG.  1    is a schematic of a bacterial monitoring system according to the present invention where a diluted bacteria culture with desired antibiotic may be aliquoted into individual well on a well plate and a sensor in the well communicates wirelessly with an impedance analyzer. 
         FIG.  2    is a schematic of exemplary IDC for use with the present invention along with its parameters, where N is the number of digits in total, L is the length of each digit, D is the gap between neighboring digits, and W is the width of the digits. 
         FIG.  3    is a COMSOL simulation of magnetic coupling between the receiver coil and the sensor coil across a piece of polystyrene plastic. 
         FIG.  4    is a graph of the frequency sweep data before and after the sensor is coupled to the receiver coil, where the yellow line represents the absolute impedance of the system and the blue line represent the phase shift. 
         FIG.  5    is a schematic of a resonant circuit design for use with the present invention, where the inductors represent the coils and the parallel resistor R1 and capacitor C1 represents the IDC on the sensor side, with Z sensor  representing the total impedance of the coil and the IDC. At the scanner side, Z int  is the intrinsic impedance of the coil, Z total  is the total measured impedance of the coil, and a signal generator/analyzer. 
         FIG.  6    is a schematic of the experimental setup showing a close up of the coated sensor as well as the finished sensor and exemplary well from a 96 well plate assembly. 
         FIG.  7    is a graph of the overnight growth curve of  E. coli  MG1655 ASV strain obtained with initial sensor design. 
         FIG.  8    is a bar chart of the method of Morris multivariable sensitivity analysis of Equation 5 where d out  is the outer diameter of the sensor. 
         FIG.  9    is a graph of a growth curve of  E. coli  MG1655 in LB and low salt LB measured by traditional OD 600  method on a Biotek Epoch2 microplate spectrophotometer over 12 hours with 15 mins interval. 
         FIG.  10    is a growth curve of  E. coli  MG1655 in low salt LB measured in the sensor system over 4 hours with 5 mins interval. 
         FIG.  11    shows growth curves of  E. coli  MG1655 , S. aureus  ALC2085, and  P. aeruginosa  PAO1 in the sensor system over 3.5 hours with 5 mins interval. 
         FIG.  12    shows growth curves of  E. coli  MG1655 and  E. coli  MG1655 ASV ampicillin resistant strain with and without 100 μg/mL ampicillin treatment. 
         FIG.  13    is a bar chart of the slopes of permittivity curves with ampicillin and ofloxacin treatment on  E. coli  MG1655 WT and untreated control. 
         FIG.  14    is a graph of the OD 600  measurement of  E. coli  MG1655 WT dose dependent response toward ampicillin. 
         FIG.  15    is a graph of the OD 600  measurement of  E. coli  MG1655 WT dose dependent response toward ofloxacin. 
         FIG.  16    is a bar chart of the slopes of permittivity curves with vancomycin and ampicillin treatment on  S. aureus  ALC2085. 
         FIG.  17    is a graph of the OD 600  measurement of  S. aureus  ALC2085 dose dependent response toward vancomycin. 
         FIG.  18    is a graph of the OD 600  measurement of  S. aureus  ALC2085 dose dependent response toward ampicillin. 
         FIG.  19    is a bar chart of the slopes of ciprofloxacin and tobramycin treatment on  P. aeruginosa  PAO1. 
         FIG.  20    is a graph of the OD 600  measurement of  P. aeruginosa  PAO1 dose dependent response toward ciprofloxacin. 
         FIG.  21    is a graph of the OD 600  measurement of  P. aeruginosa  PAO1 dose dependent response toward tobramycin. 
         FIG.  22    is a bar chart of the slopes of FBS dosed low salt LB and that of  E. coli  MG1655 grown in FBS dosed low salt LB. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Referring to the drawings, wherein like numeral refer to like parts throughout, there is seen in  FIG.  1    a system  10  for bacterial treatment monitoring according to the present invention. System  10  includes a well plate  12  having a sensor  14  positioned therein. A receiver coil  16  is positioned on the bottom of well plate  12  and wirelessly coupled to the sensor  14 . An impedance analyzer  18  is coupled to receiver coil  16  and configured to identify and record the resonance frequency of sensor  14  and receiver coil  16  over a first predetermined series of time periods. Impedance analyzer  18  is further configured to calculate the permittivity of a bacterial culture  20  placed in well plate  12 . The permittivity is tracked over a second predetermined period of time and plotted so that the slope of the plot may be used to generate a sensitivity score representing the sensitivity of bacterial culture  20  to a given antibiotic added to well plate  12  at the outset. Well plate  16  may comprise a standard 96 well plate for simultaneous assessment of multiple populations, and impedance analyzer  18  may comprise a Keysight E4990A-20 impedance analyzer. The first predetermined series of time periods may comprise five minutes, while the second predetermined period of time may comprise 30 minutes. A baseline of the sensitivity score may be determined by acquiring the growth slope of sterile LB media with treatment and known sensitive strain under treatment. The system of the present invention may thus be provided at a low cost and does not requires any special sample handling. Samples can also be loaded into standardized test plates using automated workflow systems, with report generated within 30 minutes. 
     Example 
     Bacterial Media, Reagents and Materials 
     Low salt LB medium was prepared using 0.5 g/L NaCl, 10 g/L Tryptone, 5 g/L Yeast Extract, with additions of Fetal Bovine Serum. Antibiotics tested include ampicillin, ofloxacin, ciprofloxacin, vancomycin, and tobramycin. The sensors and the receiver plate were fabricated on a flexible Polyimide Flex circuit board (Custom ordered from PCBWay Prototype to volume production Factories, Shenzhen, China). Oil-based polyurethane protective spray coating was obtained from MINWAX. Permittivity data was measured using an impedance analyzer (E4990A-20 Keysight, Santa Rosa, Calif.). 
     Bacteria Samples Preparation 
     Overnight bacterial cultures were grown in 25 mL low salt LB medium and incubated at 37° C. for 16 hours with shaking at 200 rpm. To test antibiotic susceptibility, overnight cultures were used to inoculate in a low salt LB medium with starting OD 600  of 0.001. Three hundred μL of the inoculant was then aliquoted to a 96 well plate with LC sensor inserts and quickly transferred to a 30° C. culture room for growth tracking. 
     In order to calculate the complex permittivity of the bacterial culture, an equation can be derived from parameters of the LC sensor and the resonant frequencies collected from the impedance analyzer 19-21 : 
     
       
         
           
             
               
                 
                   ε 
                   = 
                   
                     
                       
                         C 
                         1 
                       
                       
                         k 
                         ⁢ 
                         
                           ε 
                           0 
                         
                       
                     
                     - 
                     
                       ε 
                       sub 
                     
                     + 
                     
                       1 
                       
                         
                           kR 
                           1 
                         
                         ⁢ 
                         
                           ω 
                           
                             zero 
                             - 
                             inductance 
                           
                         
                         ⁢ 
                         
                           ε 
                           0 
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   1 
                   ) 
                 
               
             
           
         
       
     
     In which k is the cell constant of the IDC 22  defined by: 
     
       
         
           
             
               
                 
                   k 
                   = 
                   
                     
                       
                         L 
                         ⁡ 
                         ( 
                         
                           N 
                           - 
                           1 
                         
                         ) 
                       
                       ⁢ 
                       
                         
                           k 
                           [ 
                           
                             1 
                             - 
                             
                               
                                 ( 
                                 
                                   D 
                                   
                                     D 
                                     + 
                                     W 
                                   
                                 
                                 ) 
                               
                               2 
                             
                           
                           ] 
                         
                         
                           1 
                           2 
                         
                       
                     
                     
                       2 
                       ⁢ 
                       
                         K 
                         [ 
                         
                           D 
                           
                             D 
                             + 
                             W 
                           
                         
                         ] 
                       
                     
                   
                 
               
               
                 
                   ( 
                   2 
                   ) 
                 
               
             
           
         
       
     
     All parameters needed to calculate k can be found illustrated in  FIG.  2   , except for the elliptic integral of the first kind K[A]. Using the above permittivity equation, a permittivity vs time plot was generated. Thus, the permittivity changes in the system can be tracked, to monitor bacterial growth, allowing rapid detection of antibiotic susceptibility. 
     Signal Processing 
     The resonant circuit consists of two sides, including a circuit with a coil and an interdigitated capacitor (IDC) on the sensor side and a detection coil and a signal generator/analyzer on the scanner side, as seen in  FIG.  5   . Circuit analysis could be used to obtain the following equation from this circuit: 
     
       
         
           
             
               
                 
                   
                     Z 
                     total 
                   
                   = 
                   
                     
                       Z 
                       int 
                     
                     + 
                     
                       
                         
                           ω 
                           2 
                         
                         ⁢ 
                         
                           M 
                           2 
                         
                       
                       
                         z 
                         sensor 
                       
                     
                   
                 
               
               
                 
                   ( 
                   3 
                   ) 
                 
               
             
           
         
       
     
     In this equation, Z int  is considered as background impedance of the system and is subtracted using the built-in function of the EIS, and Z sensor  can be represented using the frequency domain as: 
     
       
         
           
             
               
                 
                   
                     Z 
                     sensor 
                   
                   = 
                   
                     
                       j 
                       ⁢ 
                       ω 
                       ⁢ 
                       
                         L 
                         2 
                       
                     
                     + 
                     
                       
                         R 
                         1 
                       
                       
                         1 
                         + 
                         
                           j 
                           ⁢ 
                           ω 
                           ⁢ 
                           
                             R 
                             1 
                           
                           ⁢ 
                           
                             C 
                             1 
                           
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   4 
                   ) 
                 
               
             
           
         
       
     
     Substituting equation (4) into equation (3), and combining with the subtraction of background noise mentioned above, the representation of total impedance on the scanner side becomes: 
     
       
         
           
             
               
                 
                   
                     Z 
                     Scanner 
                   
                   = 
                   
                     
                       
                         
                           ω 
                           2 
                         
                         ⁢ 
                         
                           M 
                           2 
                         
                         ⁢ 
                         
                           R 
                           1 
                         
                       
                       
                         
                           
                             ( 
                             
                               
                                 R 
                                 1 
                               
                               - 
                               
                                 
                                   ω 
                                   2 
                                 
                                 ⁢ 
                                 
                                   R 
                                   1 
                                 
                                 ⁢ 
                                 
                                   C 
                                   1 
                                 
                                 ⁢ 
                                 
                                   L 
                                   2 
                                 
                               
                             
                             ) 
                           
                           2 
                         
                         + 
                         
                           
                             ω 
                             2 
                           
                           ⁢ 
                           
                             L 
                             2 
                             2 
                           
                         
                       
                     
                     + 
                     
                       j 
                       ⁢ 
                       
                         
                           
                             ω 
                             2 
                           
                           ⁢ 
                           
                             
                               M 
                               2 
                             
                             ( 
                             
                               
                                 ω 
                                 ⁢ 
                                 
                                   R 
                                   1 
                                 
                                 ⁢ 
                                 
                                   
                                     C 
                                     1 
                                   
                                   ( 
                                   
                                     
                                       R 
                                       1 
                                     
                                     - 
                                     
                                       
                                         ω 
                                         2 
                                       
                                       ⁢ 
                                       
                                         R 
                                         1 
                                       
                                       ⁢ 
                                       
                                         C 
                                         1 
                                       
                                       ⁢ 
                                       
                                         L 
                                         2 
                                       
                                     
                                   
                                   ) 
                                 
                               
                               - 
                               
                                 ω 
                                 ⁢ 
                                 
                                   L 
                                   2 
                                 
                               
                             
                           
                         
                         
                           
                             
                               ( 
                               
                                 
                                   R 
                                   1 
                                 
                                 - 
                                 
                                   
                                     ω 
                                     2 
                                   
                                   ⁢ 
                                   
                                     R 
                                     1 
                                   
                                   ⁢ 
                                   
                                     C 
                                     1 
                                   
                                   ⁢ 
                                   
                                     L 
                                     2 
                                   
                                 
                               
                               ) 
                             
                             2 
                           
                           + 
                           
                             
                               ω 
                               2 
                             
                             ⁢ 
                             
                               L 
                               2 
                               2 
                             
                           
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   5 
                   ) 
                 
               
             
           
         
       
     
     It is important to note that in this equation, ω (frequency), M (mutual inductance), and L 2  (inductance of sensor coil) are all known parameters and can be controlled either by changing the input or the design of the IDC. This means only two parameters, the R 1  and C 1  are unknown and can be solved by using equation 5 after setting the imaginary part of the impedance to 0 at the zero-reactance frequency (Eq. 6) and taking the derivative of the real part of the equation (3) to set ω to resonant frequency (Eq. 7). 
     
       
         
           
             
               
                 
                   
                     ω 
                     
                       zero 
                       - 
                       inductance 
                     
                   
                   = 
                   
                     
                       
                         1 
                         LC 
                       
                       - 
                       
                         1 
                         
                           
                             R 
                             2 
                           
                           ⁢ 
                           
                             C 
                             2 
                           
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   6 
                   ) 
                 
               
             
           
         
       
       
         
           
             
               
                 
                   
                     ω 
                     resonant 
                   
                   = 
                   
                     
                       1 
                       LC 
                     
                   
                 
               
               
                 
                   ( 
                   7 
                   ) 
                 
               
             
           
         
       
     
     With these two frequencies calculated, the complex permittivity of the IDC can be calculated with the two equations mentioned above (Eq. 1 &amp; 2). 
     Electromagnetic Coupling Analysis of the Sensor 
     Two identical coils both with 0.016 mm wire thickness, 0.016 mm wire gap, 50 turns with 25 turns on each side of the polyimide flex PCB were brought together within 1.2 mm distance separated by a 0.9 mm thick polystyrene plastic well bottom of a standard 96 well plate. Power was supplied to the receiver coil by the impedance analyzer at 50 μA current level at frequencies ranging from 1 MHz to 12 MHz.  FIG.  3    shows the COMSOL simulation of the magnetic coupling of the two coils. The frequency sweep was performed with the impedance analyzer at a resolution of 1600 points, the absolute impedance and phase shift of the system were recorded before and after the sensor was brought within the coupling range of the two coils, as seen in  FIG.  4   . The result shows that the polystyrene 96 well plate is magnetically transparent enough to have negligible effect on the coupling of the two coils. 
     Construction of the Sensor System 
     Bacterial cultures were diluted and aliquoted into a standard 96 well plate with a sensor inserted in each well. A receiver coil connected to a Keysight E4990A-20 impedance analyzer on the bottom of the 96 well plates wirelessly communicate with the sensor and scans a spectrum of electrical wavelength to identify the resonance frequency between the sensor and the receiver coil. The resonance frequency of the system was recorded every 5 mins, combined with the parameter of the sensor to calculate the permittivity of the bacterial culture. The permittivity readout is plotted as a time series, and the slope of the curve over the initial 30 mins was used to access the sensitivity score of bacteria to each tested antibiotic. The baseline of the sensitivity score is determined by acquiring the time sequence slope in sterile LB medium without treatment and known sensitive strain. 
     All experiments were performed with cultures incubated at 30° C. and inoculation OD 600  of 0.001 unless noted otherwise. The positive and negative ends of the receiver coil situated in a die-cast aluminum electromagnetic insulated box is connected to the Impedance Analyzer via a pair of twisted and insulated stranded copper wire. The sensor was first mounted onto double-sided adhesive sheet and had the single layer protective polyurethane spray coating applied. The entire sheet of coated sensors was then left in a desiccator for 48 hours to ensure complete evaporation of solvent and curing of the coating material. Individual sensors were then removed from the sheet with adhesive backing, in turn rolled up as cup sleeves and placed inside of the wells, to avoid interference from protein and bacteria settling effect in a static culture environment, the sensing component of the sensor is placed vertical to the bottom of the well lining the wall of the well. Finally, the entire 96 well plate was placed in a UV Clave ultraviolet sterilization chamber for a 1-hour sterilization cycle. Three sensors were randomly selected to establish a baseline for each batch of sensors to ensure successful coating. On the Impedance Analyzer platform, a MATLAB program is used to trigger the equipment every 5 mins for a 35 mins duration, the program then takes the readout, search for the resonance frequency, and saved as time series for further analysis. The file triggered by the MATLAB program is written in VBScript to interface with the Impedance Analyzer to perform a preset 1600 points sweep within a 1 MHz range near the initial resonance frequency. 
     Sensor Design 
     Initial iteration of the sensor design composed of a fiber glass printed circuit board with 5 turns of coil on both sides of the PCB with 11 0.5 mm digits spaced 0.5 mm apart. The sensor was placed inside of a upside down 50 ml conical tubing with 20 mL of LB inoculated with 0.0001 OD600  E. coli  MG1655 ASV. The assembly is then placed on a shaker set to 200 rpm overnight in a 37° C. culture room. The growth curve against background curve obtained from the system is shown in  FIG.  7   , the curve displays lag phage, exponential growth phase, and stationary phage. However, the slower detection speed of the sensor was unsatisfactory, which motivated a redesign of the sensor design aiming to achieve faster detection speed. 
     After performing analysis of the equation with method of Morris sensitivity analysis in SALib (Sensitivity Analysis Library in Python), it was revealed that the number of turns in the coil had the largest influence on the permittivity value, followed by the outer diameter of the coil and the distance D between the digits as seen in  FIG.  8   . This was taken into consideration when designing the sensors to achieve the highest sensitivity. After several iterations of sensor design, the final design of the sensor mentioned in the method section shows the highest sensitivity with rapid detection capability. 
     Bacterial Growth Monitoring and ASTs 
     To ensure the conductive nature of the media doesn&#39;t interfere with the sensor, low salt LB was selected as the medium for bacterial growth in this study. Growth curves for both regular LB and low salt LB were recorded with traditional OD600 measurement over a period of 12 hours ( FIG.  9   ). Baseline readout of sterile medium was established and subsequently subtracted as background from the experimental data below. Initial growth monitoring was tested with  E. coli  MG1655, as seen in  FIG.  10   . Data revealed that compared to the growth curve based on OD 600 , relative permittivity was more sensitive in detecting bacterial growth. Upon inoculation, there was an immediate increase in permittivity, likely due to nonspecific adsorption of proteins in growth medium onto the polyurethane coating. The protein adsorbed onto the sensor surface quickly saturates and forms a protein shell that is tens of nanometers thick, and this is observed in the sterile medium permittivity curve. Similar phenomena were observed when measuring the baseline, where there was an initial moderate increase in signal for sterile media alone. Further increase due to bacterial growth was recorded around 30 mins after inoculation, much earlier than OD600 measurement (around 5 h). The growth curve collected by the system showed logarithmic increase in relative permittivity which then slowed down over time and eventually reached a plateau. The presence of plateau is presumably due to the saturation of free space on the sensor surface by bacterial growth and associated products. Further experiments were done on two more different strains of both Gram-positive and Gram-negative bacteria to test if the functionality of the sensor extends beyond just  E. coli . As shown in  FIG.  11   , not only do all three strains elicit similar responses over the 3.5 hours monitoring period, the three strains show some segments of different patterns of growth, which can be investigated in future research for potential of strain identification using specific medium. 
     To determine if this sensor can be used for AST, an ampicillin sensitive strain  E. coli  MG1655 and ampicillin resistant strain  E. coli  MG1655 ASV were compared for growth in the absence and presence of ampicillin. Both strains were dosed with 100 μg/mL ampicillin at the time of inoculation and cultured for 4 hours, which is known to induce cell lysis in sensitive  E. coli  strains. As  FIG.  12    shows,  E. coli  ASV strain grew in presence of ampicillin while the wildtype  E. coli  MG1655 strain was inhibited by the ampicillin treatment as expected, all of which was observed within 30 mins. The low initial increase in relative permittivity maybe from bacteria in the suspension being lysed, and the cellular content increased the permittivity near the sensor surface. 
     Subsequent experiments were performed on more antibiotics and all three previously tested bacterial species, with the detection window of sensitivity to antibiotics within 30 mins. In order to create a more uniform test time towards the goal of creating a standard method, the test time was set universally at 30 mins with 5 mins read interval, since most growth curves shows initial signs of resistance or growth in that time frame, as seen in  FIG.  12   . At the same time, to obtain more rapid detection of growth and distinguish resistant vs. sensitive strains, a linear regression was opted in on the relative permittivity on a logarithmic scale. The slopes of the best fit line were then used as a sensitivity score to determine the sensitivity of strains toward antibiotics. It can be seen in  FIGS.  13 - 21    that the dose dependency nature of bacterial susceptibility to antibiotics can be seen in both LC sensor system and OD600 measurement, where trends of growth inhibition after 6-10 hours in OD600 measurement was captured by the slope of permittivity measurement substantially earlier in just 30 mins. 
     Performance in the Presence of Host Proteins 
     Samples collected in a clinical setting is often complex. Whole blood, plasma, serum, urine, or other liquid from the human body contains significantly more types and larger amounts of proteins and cells compared to a laboratory culture media. Thus, it is necessary to test the sensor system with the addition of complex components in order to determine the feasibility of implementing this system in clinical setting where sample preparation beyond dilution and centrifugation is seldom performed. For the above reasons, fetal bovine serum was selected to mimic human serum and added to low salt LB at different percentages. The response of the sensor system is shown in  FIG.  22   . Encouragingly, at lower concentrations, there is 1 sufficient free space near the surface of the sensor for sensing bacterial growth. For low salt LB dosed with 2% of FBS, the growth of  E. coli  wildtype strain MG1655?? was captured by the LC sensor system with little interference from the serum. This demonstrates the possibility of using these sensors in clinical settings. When the amount of PBS reached 5%, the response was lost, likely because the free space thickness was saturated by protein adsorption. 
     
       
         
           
             
               
                 
                   
                     d 
                     sat 
                   
                   = 
                   
                     
                       - 
                       
                         D 
                         
                           a 
                           1 
                         
                       
                     
                     ⁢ 
                     
                       ln 
                       ⁡ 
                       ( 
                       
                         0.005 
                         
                           a 
                           2 
                         
                       
                       ) 
                     
                   
                 
               
               
                 
                   ( 
                   8 
                   ) 
                 
               
             
           
         
       
       
         
           
             
               
                 
                   
                     a 
                     1 
                   
                   = 
                   
                     
                       114.97 
                       
                         
                           ( 
                           
                             W 
                             + 
                             D 
                           
                           ) 
                         
                         3 
                       
                     
                     + 
                     
                       28.75 
                       
                         
                           ( 
                           
                             W 
                             + 
                             D 
                           
                           ) 
                         
                         2 
                       
                     
                     - 
                     
                       9.183 
                       
                         ( 
                         
                           W 
                           + 
                           D 
                         
                         ) 
                       
                     
                     + 
                     1.631 
                   
                 
               
               
                 
                   ( 
                   9 
                   ) 
                 
               
             
           
         
       
       
         
           
             
               
                 
                   
                     a 
                     2 
                   
                   = 
                   
                     
                       1293.21 
                       
                         
                           ( 
                           
                             W 
                             + 
                             D 
                           
                           ) 
                         
                         3 
                       
                     
                     + 
                     
                       164.87 
                       
                         
                           ( 
                           
                             W 
                             + 
                             D 
                           
                           ) 
                         
                         2 
                       
                     
                     - 
                     
                       6.521 
                       
                         ( 
                         
                           W 
                           + 
                           D 
                         
                         ) 
                       
                     
                     + 
                     6.105 
                   
                 
               
               
                 
                   ( 
                   10 
                   ) 
                 
               
             
           
         
       
     
     With thicker free space for sensing, the system can be further optimized to capture more complex samples, and longer-term growth pattern. 
     This study demonstrated that the feasibility of rapid AST using LC sensor in a 96 well plate format. To our best knowledge, this is the fastest AST test at the phenotype level without using complex equipment. Compared to genotypic ASTs, this system only requires simple sample preparation (dilution only) and can be fitted into an automated workflow for high-throughput detection. The system can generate reports with little computational power without needing an advanced data analyst or the capability of handling massive data sets (e.g., those required by MALDI-TOF). The system is also not limited to the use of specific cartridges for a limited selection of strains, instead uses a reference library for identifying MIC of antibiotics of interest. Compared with other existing automated phenotypical approaches, this method achieves AST with significantly shorter detection time (˜30 mins). The system can be optimized in the future by employing high-throughput method in which the whole 96 well-plate can be processed in a relatively short amount of time, enabling a more comprehensive assay. This is part of our ongoing work. The coating material and coating thickness of the chips can also be optimized to achieve higher accuracy and tolerance to fouling from the growth media or cellular products. The results of 2% FBS demonstrates the possibility to test clinical samples after simple dilution of patient sample. To handle even higher serum percentage, the IDE design could be adjusted to increase the saturation thickness ergo increase the upper concentration limit of FBS content. It has been reported that the saturation thickness can be described by Eq. 8-10, where a1 and a2 are both functions of digit width W and gap width D. 
     
       
         
           
             
               
                 
                   
                     d 
                     sat 
                   
                   = 
                   
                     
                       - 
                       
                         D 
                         
                           a 
                           1 
                         
                       
                     
                     ⁢ 
                     
                       ln 
                       ⁡ 
                       ( 
                       
                         0.005 
                         
                           a 
                           2 
                         
                       
                       ) 
                     
                   
                 
               
               
                 
                   ( 
                   8 
                   ) 
                 
               
             
           
         
       
       
         
           
             
               
                 
                   
                     a 
                     1 
                   
                   = 
                   
                     
                       114.97 
                       
                         
                           ( 
                           
                             W 
                             + 
                             D 
                           
                           ) 
                         
                         3 
                       
                     
                     + 
                     
                       28.75 
                       
                         
                           ( 
                           
                             W 
                             + 
                             D 
                           
                           ) 
                         
                         2 
                       
                     
                     - 
                     
                       9.183 
                       
                         ( 
                         
                           W 
                           + 
                           D 
                         
                         ) 
                       
                     
                     + 
                     1.631 
                   
                 
               
               
                 
                   ( 
                   9 
                   ) 
                 
               
             
           
         
       
       
         
           
             
               
                 
                   
                     a 
                     2 
                   
                   = 
                   
                     
                       1293.21 
                       
                         
                           ( 
                           
                             W 
                             + 
                             D 
                           
                           ) 
                         
                         3 
                       
                     
                     + 
                     
                       164.87 
                       
                         
                           ( 
                           
                             W 
                             + 
                             D 
                           
                           ) 
                         
                         2 
                       
                     
                     - 
                     
                       6.521 
                       
                         ( 
                         
                           W 
                           + 
                           D 
                         
                         ) 
                       
                     
                     + 
                     6.105 
                   
                 
               
               
                 
                   ( 
                   10 
                   ) 
                 
               
             
           
         
       
     
     With thicker free space for sensing, the system can be further optimized to capture more complex samples, and longer-term growth pattern. The design of the LC sensor too, can be modified and optimized to fit into various format. For example, lithography could be used to fit the system into smaller format or microfluidic devices.