TW202219276A - Microfluidic chips with plasmonic sensors - Google Patents

Abstract

In example implementations, a microfluidic chip is provided. The microfluidic chip includes a first channel, a second channel, and a plasmonic sensor. The first channel includes a first antibiotic chamber and a first filter chamber. The first antibiotic chamber includes a first media and a first antibiotic that is rehydrated by a bacterial suspension and the first filter chamber is to filter bacteria from first metabolites released from the bacteria in response to incubation with a buffer solution. The second channel includes a second antibiotic chamber and a second filter chamber. The second antibiotic chamber includes a second media and a second antibiotic that is rehydrated by the bacterial suspension and the second filter chamber is to filter the bacteria from second metabolites released from the bacteria in response to incubation with the buffer solution. The first channel and the second channel pass over the plasmonic sensor. The plasmonic sensor is to measure an amount of the first metabolites and the second metabolites.

Description

Microfluidic chip with plasmonic sensor

The present invention relates to microfluidic chips with plasmonic sensors.

Antibiotic susceptibility testing (AST) can be used to analyze the effectiveness of antibiotics against specific strains of bacteria. AST can be used for appropriate and effective prescription of antibiotics and doses to be administered to patients. AST can analyze the effectiveness of antibiotics by measuring the amount of bacteria in a solution with antibiotics. For example, the bacteria can be mixed with a nutrient medium that allows the bacteria to grow. Nutrient media can be replaced with water, which causes the bacteria to undergo metabolic changes in response to the absence of nutrients. To persist in this environment, bacterial cells can initiate a series of dissimilatory reactions and by-products of these processes can be released purine metabolites. Specific strains of bacteria can be assayed for the presence of purine metabolites.

In some examples, bacteria can be grown with a nutrient medium mixed with antibiotics and then transferred to water. Changes in the amount of purine metabolites in response to antibiotics can be analyzed to determine the effectiveness of antibiotics.

Some AST methods have turnaround times that can be hours. For example, the turnaround time may be 12-24 hours. These methodologies may also include bacterial culture and isolation steps prior to AST, which may have additional processing time of 18-24 hours.

In one aspect of the present invention, a microfluidic chip is disclosed, comprising: a first channel comprising a first antibiotic chamber and a first filtration chamber, wherein the first antibiotic chamber comprises a first culture medium with a first antibiotic, which is rehydrated by a bacterial suspension, and the first filter chamber is used to filter separate bacteria from a first metabolite released by the bacteria in response to incubation with a buffer solution; a second channel comprising a second antibiotic chamber and a second filter chamber, wherein the second antibiotic chamber comprises a second culture medium and a second antibiotic, which is rehydrated by the bacterial suspension, and the second filter a chamber for filtering separating bacteria from a second metabolite released by the bacteria in response to incubation with the buffer solution; and a plasmonic sensor, wherein the first channel and the second channel pass through the plasmon Above the sub-sensor, wherein the plasmonic sensor is used to measure the amount of the first metabolite and the second metabolite.

The examples described herein provide a microfluidic chip with a plasmonic sensor for performing the antibiotic susceptibility test (AST) of the present disclosure. As noted above, AST can be used to analyze the effectiveness of antibiotics against a particular strain of bacteria. However, current methodologies may have a turnaround time of several hours (eg, 12-24 hours).

However, due to these long turnaround times, most patients are prescribed a broad-spectrum antibiotic when an infection is suspected. These broad-spectrum prescriptions are sometimes completely ineffective because of the spread of antibiotic-resistant bacterial strains. In these cases, the patient's condition may rapidly regress and the chance of life-threatening complications may increase over the hours.

The present disclosure provides a microfluidic chip with a plasmonic sensor that can reduce the turnaround time of AST to minutes to hours (eg, less than 2 hours). The microfluidic chips of the present disclosure are relatively low-cost, highly accurate, and can be scaled up for large-scale distribution. In addition, microfluidic chips with plasmonic sensors can analyze multiple different concentrations or types of antibiotics against a bacterial strain within a single microfluidic chip. Thus, the present disclosure may reduce the turnaround time of AST, which may allow an infected patient to be prescribed an appropriate dose of a particular antibiotic.

1 illustrates a block diagram of an example microfluidic chip of a plasmonic sensor 100 (also referred to herein as a chip 100) of the present disclosure. In one example, the wafer 100 may include an inlet 102 , channels 104 ₁ and 104 ₂ , and a plasmonic sensor 120 . Although two channels 104 ₁ and 104 ₂ are illustrated in FIG. 1 , it should be noted that any number of channels 104 may be deployed. Examples of how wafer 100 may be deployed or fabricated are discussed in further detail below with reference to FIGS. 2-6 . Although a single inlet 102 is illustrated in FIG. 1 , it should be noted that several separate inlets 102 may also be deployed for the channel 104 .

In one example, the channel 104 ₁ may include a first antibiotic chamber 106 ₁ and a first filtration chamber 110 ₁ . Although shown as separate chambers, in some implementations, the first antibiotic chamber 1061 and the _first filtration chamber ₁₁₀₁ may be combined into a single chamber. The first antibiotic chamber 106 ₁ may include a first antibiotic or a first dose of antibiotic, and a culture medium 108 ₁ (hereinafter also simply referred to as culture medium 108 ₁ ). Medium 108 ₁ may be a lyophilized mixture of antibiotic and medium. In _one example, the culture medium 1081 may include nutrients that aid bacterial growth, as discussed in further detail below. Examples of medium 1081 may include Luria _- Bettany (LB) broth, tryptic soy broth, nutrient broth, and the like.

In one example, the culture medium 108 ₁ may be dispensed into the first antibiotic chamber 106 ₁ when the wafer 100 is fabricated. The medium 1081 may be prepared during wafer ₁₀₀ fabrication and dispensed via a freeze drying method or an ink jet printing method.

The first filter chamber 110 ₁ may include a filter 112 ₁ . Filter 1121 may be used to filter out metabolites 116 produced by bacteria 114 in _first filtration chamber ₁₁₀₁ . Metabolites 116 can be filtered out of the first filter chamber 110 ₁ , through a first outlet 122 ₁ and above the plasmonic sensor 120 .

In one example, the channel 104 ₂ may include a second antibiotic chamber 106 ₂ and a second filter chamber 110 ₂ . Although shown as separate chambers, in some implementations, the second antibiotic chamber 1062 and the _second filtration chamber ₁₁₀₂ may be combined into a single chamber. The _second antibiotic chamber 1062 may include a second antibiotic or a second dose of antibiotic, and culture medium ₁₀₈₂ (hereinafter also simply referred to as culture medium ₁₀₈₂ ). Similar to medium 108 ₁ , medium 108 ₂ may be a lyophilized mixture of antibiotic and medium. In one example, the culture medium ₁₀₈₂ may include nutrients that aid bacterial growth, as discussed in further detail below. Examples of medium 1082 may include Luria-Bettany (LB) broth, tryptic soy broth, nutrient broth, and the like.

In one example, the culture medium 108 ₂ may be dispensed into the second antibiotic chamber 106 ₂ when the wafer 100 is fabricated. The medium ₁₀₈₂ may be prepared during wafer 100 fabrication and dispensed via a freeze drying method or an ink jet printing method.

The second filter chamber 110 ₂ may include a filter 112 ₂ . Filter 1122 may be used to filter out metabolites 116 produced by bacteria 114 in _second filtration chamber ₁₁₀₂ . Metabolites 116 may be filtered out of the second filtration chamber 110 ₂ , through the second outlet 122 ₂ and above the plasmonic sensor 120 .

In one example, a bacterial suspension may be introduced into wafer 100 via inlet 102 . The bacterial suspension can be controlled to flow into the first antibiotic chamber 106 ₁ and the second antibiotic chamber 106 ₂ . The bacterial suspension can be rehydrated with media 108 ₁ and 108 ₂ , respectively. After an incubation period (eg, several minutes), the mixture in the first antibiotic chamber 106 ₁ and the second antibiotic chamber 106 ₂ can move into the first filtration chamber 110 ₁ and the second filtration chamber 110 ₂ .

In one example, a buffer solution may be introduced into wafer 100 via inlet 102 (or a separate inlet as indicated above). The buffer solution, which can be water or another fluid, is used to rinse the bacteria in the bacterial suspension and remove the culture medium and antibiotics _108i and ₁₀₈₂ , and by microorganisms in the _first filtration chamber 1101 and the second filtration chamber Metabolites released in 110 ₂ .

With the media 1081 and 1082 removed, the bacteria 114 _remaining in the _first filtration chamber ₁₁₀₁ and the _second filtration chamber 1102 can be allowed to grow to induce a stress response. Bacteria 114 may release stress-induced metabolites 116 in response to stress. Metabolite 116 may flow through filters 112 ₁ and 112 ₂ , respectively, and toward plasmonic sensor 120 .

In one example, the filters 112 ₁ and 112 ₂ may be porous filter membranes. In another example, the filters 112 ₁ and 112 ₂ may include physical structures that allow the metabolites 116 to pass through while retaining the bacteria 114 . For example, the structures may include upstanding posts. In one example, microbeads of different sizes with reduced diameters can be loaded between the pillars to achieve a desired porosity.

_Plasmonic sensor ₁₂₀ can be used with an optical sensor to measure the amount of metabolite 116 in outlet channels 1221 and 1222. In one example, the plasmonic sensor 120 may be a surface-enhanced Raman spectroscopy (SERS) sensor, a surface-enhanced infrared absorption (SEIRA) sensor, a surface-enhanced fluorescence (SEF) sensor devices, surface-enhanced luminescence (SEL), and the like. The light sources may emit light onto exit channels ₁₂₂₁ and ₁₂₂₂ to induce detection. The light or beam can be scattered by the plasmonic sensor 120 and read by an optical detector or sensor. The scattered light can be converted by an optical detector into an image or map, which can correspond to the amount of metabolite 116 in outlets ₁₂₂₁ and ₁₂₂₂ .

Different measurements can determine which antibiotic or antibiotic dose is more effective against bacteria 114 in the _first antibiotic chamber 1061 and the _second antibiotic chamber 1062. Thus, an appropriate antibiotic and an appropriate antibiotic dosage can be prescribed based on the analysis performed using the chip 100 .

In one example, the wafer 100 may also include a temperature controller (not shown). A temperature controller may be used to control the temperature of the antibiotic chambers 106 ₁ and 106 ₂ and/or the filtration chambers 110 ₁ and 110 ₂ . The temperature controller can help simulate the natural growth environment of the bacteria 114 or accelerate the production of metabolites 116 derived from the stress-induced response of the bacteria 114 .

In one example, the temperature controller may be external. For example, wafer 100 may be held in a temperature-controlled space, such as an incubator. In one example, the temperature controller may be part of the wafer. For example, the temperature controller may include a resistor and a temperature sensor (eg, a thermistor) on the substrate of the chip 100 . In one example, the temperature controller may be a thin film resistor.

2 illustrates an example of a microfluidic chip having a plasmonic sensor 200 (also referred to herein as a chip 200). In one example, the wafer 200 may be fabricated as a multi-layer wafer with an integrated plasmonic sensor 220 . FIG. 2 illustrates a top view of the wafer 200 .

In one example, the wafer 200 may include an inlet 201 and an inlet 202 . The inlet 201 can be used to introduce a bacterial suspension. The bacterial suspension may include a specific strain of bacteria to be analyzed. Inlet 202 can be used to introduce a buffer solution.

The wafer 200 may include a plurality of channels _204i - _204n (hereinafter also referred to individually as channels 204 or collectively as channels 204). Each channel 204 may include a respective antibiotic chamber 206 and a corresponding filter chamber 210 . Antibiotic chamber 206 may include a lyophilized mixture of an antibiotic and a culture medium 208. Culture medium 208 can be introduced into antibiotic chamber 206, similar to culture media _108i and ₁₀₈₂ in wafer 100, as discussed above.

Filtration chamber 210 may include a filter 212 . The filter 212 can be a porous membrane or a physical structure (eg, a pillar). The filter 212 may allow the passage of the metabolites 216 while blocking the bacteria 214. Metabolite 216 can flow through the channel above plasmonic sensor 220 .

In one example, the filter 212 may include a second lyophilized mixture 213 . The lyophilized mixture may include the accelerator as well as a calibration molecule. In one example, calibration molecules can be used as internal standards for more quantitative SERS measurements.

In one example, the promoter can accelerate the stress response of the bacteria 214 . Promoters may include phosphates that cause the bacteria 214 to release purine metabolites immediately or within minutes of incubation. This can also help reduce the turnaround time for performing AST in the wafer 200 . In one example, promoters may include diphosphates, phosphoribosyltransferases, nucleoside monophosphates, and the like.

In one example, each of the channels 204 may include an antibiotic chamber 206 and a filtration chamber 210 . Each antibiotic chamber 206 may contain a different antibiotic or different doses of the same antibiotic. In one example, one of the channels 204 may include reporter molecules for in-situ calibration of the optical sensor 224 . Each channel 204 can travel across the plasmonic sensor 220 .

In one example, the wafer 200 may also include capillary breaks 218 . Capillary gap 218 may allow a gas or air to pass through a channel (shown in FIG. 3 ) to control the flow of fluid within channel 204 of wafer 200 . The wafer 200 may also include an outlet 222 to allow fluid to exit the wafer 200 through vias 230 located between the layers of the wafer 200 .

In one example, the wafer 200 may also include a temperature controller (not shown). A temperature controller may be used to control the temperature of the antibiotic chamber 206 and/or the filtration chamber 210. The temperature controller can help simulate the natural growth environment of the bacteria 214 or accelerate the production of metabolites 216 derived from the stress-induced response of the bacteria 214 .

In one example, the temperature controller may be external. For example, wafer 200 may be held in a temperature-controlled space, such as an incubator. In one example, the temperature controller may be part of the wafer. For example, the temperature controller may include a resistor and a temperature sensor (eg, a thermistor) on the substrate of the chip 200 . In one example, the temperature controller may be a thin film resistor.

FIG. 3 shows an example cross-sectional side view of wafer 200 . As can be seen in FIG. 3 , the wafer 200 may include a first layer 250 , a second layer 252 and a third layer 254 . The first layer 250 may include a substrate 256 on which the plasmonic sensor 220 is fabricated. The substrate 256 may be a semiconductor (eg, a silicon substrate). The remaining layers may be formed from a photodefinable polymer (eg, SU8). Channel 204, antibiotic chamber 206, filter chamber 210, through-hole 230, and outlet 222 may be formed by a photolithography process that defines and etches features of the photodefinable polymer.

In one example, a portion of the first layer 250 may include an optically transparent layer 258 . The optically transparent layer 258 may provide a window above the plasmonic sensor 220 that allows the light beam to reach the channel 204 above the plasmonic sensor 220 .

In one example, the filter 212 may be disposed between the second layer 252 and the first layer 250 . In one example, the filter 212 may be selectively deployed at a location between the first layer 250 and the second layer 252 where the channel 204 is open. This alternatively, the filter 212 may be included in the portion of the second layer 252 that includes the filter chamber 210 .

In one example, the capillary gap 218 may be formed as a film layer between the second layer 252 and the third layer 254 . Fluid can flow across the channels 232 in the third layer 254 and across the membrane to create a vacuum. Fluid can be controlled to flow over different portions of the membrane at certain capillary breaks 218 to control the flow of fluid within channel 204 .

In one example, the membrane may comprise a nanoporous hydrophobic membrane that prevents liquid flow therethrough but allows air or gas flow therethrough. The membrane may have pores with an average diameter of about 200 to 400 nanometers (nm) and may be fabricated from polytetrafluoroethylene (PTFE). Other example materials for the film may include poly(propylene), poly(ethylene), and the like.

In one example, optical sensor 224 and light source 226 may be used to perform Raman measurements on wafer 200 . In one example, the optical sensor 224 may be a Raman spectrometer or a charge coupled device (CCD) detector that produces a CCD image. The light source 226 can be a laser light source. However, the light source 226 may have a length equal to the length of the channel 204 of the plasmonic sensor 220 across the row. Therefore, measurements can be taken from an integral portion of the span channel 204 that runs across the plasmonic sensor 220 . In one example, the light source 226 may illuminate each channel 204 across the plasmonic sensor 220 simultaneously. In one example, multiple light sources 226 may be deployed, or a single light source 226 with an optical grating may be deployed.

In one example, optical sensor 224 may include lenses, filters, diffraction gratings, and other devices (not shown) to focus incident light scattered by plasmonic sensor 220 onto a detector array . In one example, optical sensor 224 may split incident light into different channels, each of which is sent to a different sensor within optical sensor 224, provided by plasmonic sensor 220. Multispectral analysis of scattered light. Optical sensor 224 may perform brightfield, darkfield, fluorescence, hyperspectral, and other optical analyses.

FIG. 4 illustrates an example operational flow diagram of a method 400 of performing AST with the wafer 200 illustrated in FIGS. 2 and 3 . At block 402, method 400 loads a bacterial suspension via inlet 201. The bacterial suspension may include a specific bacterial strain to be examined or analyzed for an effective antibiotic or antibiotic dose. This bacterial suspension can be delivered to the antibiotic chamber 206 . For example, a fluid can flow through the capillary gap 218 of the antibiotic chamber 206 to create a vacuum that pulls the bacterial suspension into the antibiotic chamber 206 . Excess bacterial suspension can be removed through through hole 230 and outlet 222 .

At block 404, air 260 may be forced through channel 204 to purge the loading channel. Air 260 can remove any residual bacterial suspension that may remain in channel 204 .

At block 406, the bacterial suspension may rehydrate the medium 208 and allow to incubate for a predetermined period of time (eg, several minutes). The nutrients in the medium 208 can allow the bacteria to grow and the antibiotics in the medium 208 can kill the bacteria. The effectiveness of an antibiotic can be measured based on the amount of metabolite released and measured, as discussed in further detail below.

In one example, one of the channels 204 may include a control group. Each antibiotic chamber 206 may contain a different antibiotic or a different dose of antibiotic. Thus, the wafer 200 may allow simultaneous analysis of different antibiotics or doses against a particular bacterial strain with a relatively short turnaround time.

At block 408 , the outgrown bacteria may be drawn into the filter chamber 210 . For example, a fluid can flow across the capillary gap 218 of the filtration chamber 210 to create a vacuum that pulls outgrown bacteria from the antibiotic chamber 206 into the filtration chamber 210. Filtration chamber 210 may include a lyophilized mixture 213 . As described above, the lyophilized mixture 213 may include a calibration molecule and/or an accelerator.

At block 410, a buffer solution may be introduced via inlet 202. The buffer solution can be water, a solvent, or any other liquid that can be used to wash the residual medium mixed with bacteria 214 in the filtration chamber 210. Again, the capillary gap 218 in the filter chamber 210 can be used to pull in the buffer solution. Excess buffer solution can be removed through through hole 230 and outlet 222 .

At block 412, the bacteria 214 may be allowed to grow in the medium without nutrients. Incubation induces a stress response that causes bacteria 214 to release metabolite 216. In one example, the promoter from the lyophilized mixture 213 can interact with the bacteria 214 in the filtration chamber 210 to accelerate the release of the metabolite 216 immediately or within minutes.

At block 414 , metabolite 216 is transferred through filter 212 and into channel 204 above plasmonic sensor 220 . For example, fluid can flow through the capillary gap 218 across the end of the channel 204 to pull the metabolite 216 through the filter 212 and over the plasmonic sensor 220 . As discussed above, filter 212 may be a porous filter membrane or structure (eg, a column) that allows metabolites 216 to pass through filter 212, but prevents bacteria 214 from passing through filter 212.

At block 416 , air may be delivered through the inlet 202 to dry the channel 204 . The channel can be dried from any residual liquid and the remaining bacteria 214 can be removed via the outlet 222.

At block 418, a Raman measurement may be performed. As described above, a light source may direct the light beam over the plasmonic sensor 220 . Light can be scattered by plasmonic sensor 220 and detected by optical sensor 224 illustrated in FIGS. 2 and 3 .

As described above, the wafer 200 may allow for analysis of different antibiotics and/or doses. Based on this measurement, the most effective antibiotic or dose can be selected to treat the infection with a particular bacterial strain. The turnaround time for analysis with wafer 200 may be minutes to hours. Furthermore, the relatively low cost of manufacturing wafer 200 may allow wafer 200 to be scaled up for high volume production and fabrication.

5 illustrates a top view of a block diagram of another example of a microfluidic chip with a plasmonic sensor 500 (hereinafter also referred to as a chip 500). In one example, wafer 500 may be fabricated on a single layer along parallel channels 504i _{- 504n} (hereinafter also referred to individually as a channel 504 or collectively as channel 504). For example, the channel 504 can be placed on a single horizontal plane through a substrate.

In one example, the wafer 500 may include an inlet 501 , a plurality of channels 504 ₁ - 504 _n , and an outlet 530 . Although a single portal 501 is illustrated in FIG. 5, it should be noted that multiple portals 501 may be deployed.

Each channel 504 may include a respective antibiotic chamber _{506i - 506n} , and a respective filtration chamber _5101-510n . Each antibiotic chamber 506i _{- 506n} may include a respective antibiotic and medium 508i _{- 508n} . Each filter chamber 510 ₁ - 510 _n may include a respective filter 512 ₁ - 512 _n . Filter 512 may be a porous filter membrane or structure (eg, a column), as described above.

Each filter chamber 510 ₁ to 510 _n may branch into two separate outlet channels 518 and 522 . The outlet channel 518 may include a plasmonic sensor 520 . For example, filtration chamber 510 ₁ may include outlet channels 518 ₁ and 522 ₁ . Outlet channel 518 ₁ may include plasmonic sensor 520 ₁ . Filtration chamber 510 ₂ may include outlet passages 518 ₂ and 522 ₂ . Outlet channel 518 ₂ may include plasmonic sensor 520 ₂ . Filtration chamber _510n may include outlet channels _518n and _522n . The outlet channel _518n may include a plasmonic sensor _520n .

In one example, wafer 500 may include valves 504i _{- 504n} , valves 509i _{- 509n} , valves _{514i -} 514n, and valves 516i _{- 516n} . Valves 504, 514, 509, and 516 may be mechanically or electromechanically operated microfluidic valves. Valves 504 , 514 , 509 and 516 may be used to control the flow of liquids or fluids through wafer 500 and through respective channels 502 .

In one example, wafer 500 may be used with a light source and an optical sensor (not shown), similar to light source 226 and optical source 224 described above. The light source 224 can emit light onto each of the plasmonic sensors 520i _{- 520n} , and the response can be detected and measured by the optical sensor.

FIG. 6 illustrates an example operational flow diagram of a method 600 of performing AST in a wafer 500 of the present disclosure. At block 602 , the bacterial suspension can be loaded via inlet 501 . The bacterial suspension may include a specific bacterial strain to be examined or analyzed for an effective antibiotic or antibiotic dose.

In one example, valve 504 may be opened while valve 509 remains closed. The bacterial suspension can be delivered to the antibiotic chamber 506 . Antibiotic chamber 506 may include a mixture of an antibiotic and a culture medium 508 . Each antibiotic chamber 506i _{- 506n} may contain different antibiotics or different doses of the same antibiotic. In one example, one of the antibiotic chambers 506i _{- 506n} may include a control group.

At block 604, valve 504 may be closed. The bacterial suspension including bacteria 532 can rehydrate medium 508. Medium 508 may include nutrients that allow bacteria 532 to grow. Antibiotics may kill bacteria532. The effectiveness of an antibiotic or a particular dose of antibiotic can be judged based on the amount of metabolite released, which can be related to the amount of bacteria remaining, as discussed in further detail below. The bacteria 532 may be allowed to incubate for a predetermined amount of time (eg, minutes to hours).

At block 606, valves 504, 509, and 514 may be opened, and valve 516 may be closed. Buffer solutions can be delivered via inlet 501 . The buffer solution can propel bacteria 532 and culture medium 508 into filtration chamber 510 . The buffer solution can rinse the bacteria 532 to remove the culture medium 508 to exit through the valve 514 , through the outlet channel 522 , and through the outlet 530 .

At block 608, valves 514 and 516 may be closed to allow bacteria 532 to grow in filtration chamber 510 for a predefined amount of time (eg, several minutes). In the absence of nutrients in medium 508, bacteria 532 can undergo a stress response. Stress reactions can cause bacteria 532 to release metabolites 534. In one example, accelerants may be added to bacteria 532 to accelerate the release of metabolites 534.

At block 610 , valve 516 may be opened to allow metabolite 534 to flow through channel 518 . Metabolites can flow over respective plasmonic sensors 520 in channels 518 . In one example, valve 516 can be closed and valve 514 can be opened to flush remaining bacteria 532 through valve 514 and out through channel 520 .

In one example, valve 514 may be closed and valve 516 may be opened to dry outlet channel 520 . For example, air may be blown through wafer 500 to dry outlet channel 520 .

At block 612, a laser (eg, laser light source 226) may emit beams or rays over plasmonic sensors 520i _{- 520n} . Light scattered by the plasmonic sensor 520 can be read by an optical sensor. Optical sensors can measure the amount of metabolite 536 based on the scattered light signal. The amount of metabolite 536 can be correlated to the amount of bacteria, which can then be used to measure the effectiveness of antibiotics or doses within a particular antibiotic chamber 506 . The most effective antibiotic or antibiotic dose can be selected or prescribed to treat infections caused by bacteria 532.

As described above, the wafer 500 may allow analysis of different antibiotics and/or doses. Based on this measurement, the most effective antibiotic or dose can be selected to treat the infection with a particular bacterial strain. The turnaround time for analysis with wafer 500 can range from minutes to hours. Furthermore, the relatively low cost of manufacturing the wafer 500 may allow the wafer 500 to be scaled up for mass production and fabrication.

In one example, the wafer 500 may also include a temperature controller (not shown). A temperature controller may be used to control the temperature of the antibiotic chamber 506 and/or the filtration chamber 510. The temperature controller can help simulate the natural growth environment of the bacteria 532 or accelerate the production of metabolites 534 derived from the stress-induced response of the bacteria 532 .

In one example, the temperature controller may be external. For example, wafer 500 may be held in a temperature-controlled space, such as an incubator. In one example, the temperature controller may be part of the wafer. For example, the temperature controller may include a resistor and a temperature sensor (eg, a thermistor) on the substrate of the chip 500 . In one example, the temperature controller may be a thin film resistor.

FIG. 7 illustrates a top view of an example of how microfluidic channels 702 , 704 and 706 may be routed over plasmonic sensor 720 . Channels 702, 704, and 706 may be part of wafers 100, 200, or 500 illustrated in Figures 1-6 and discussed above. The plasmonic sensor 720 may be similar to the plasmonic sensors 120, 220, and 520 illustrated in Figures 1-6 and discussed above.

In one example, channels 702, 704, and 706 may be routed over plasmonic sensor 720 several times. This can provide an average reading from the optical sensor and provide a more accurate measurement. In one example, each portion of channels 702, 704, and 706 that travel over plasmonic sensor 720 may run in a straight line. In one example, channels 702 , 704 and 706 may be routed over plasmonic sensor 720 in a serpentine fashion to achieve multiple passes over plasmonic sensor 720 .

As can be seen in FIG. 7, curves or bends can occur outside the active area of plasmonic sensor 720. This linear motion can occur above the plasmonic sensor 720 . This linear operation may allow a light source emitting light in a line to be used to measure the entire portion of channels 702 , 704 and 706 above plasmonic sensor 720 .

Several lines of light can be emitted to measure each portion of each channel 702 , 704 and 706 above the plasmonic sensor 720 simultaneously. For example, in FIG. 7 , there are nine lines spanning over plasmonic sensor 720 . The light source may emit nine separate rays (eg, using optical gratings or different light sources) to measure all of the portions of channels 702, 704, and 706 covering plasmonic sensor 720 simultaneously.

It should be noted that the meandering pattern illustrated in FIG. 7 is an example pattern. Other patterns can be deployed to achieve the same result. Furthermore, although three channels are illustrated in Figure 7, any number of channels may be deployed.

8 illustrates a flow diagram of a method 800 of a method of performing AST with a microfluidic wafer incorporating the plasmonic sensor of the present disclosure. In one example, method 800 may be performed by microfluidic wafer 100 illustrated in Figure 1, microfluidic wafer 200 illustrated in Figures 2 and 3, or microfluidic wafer 500 illustrated in Figure 5, and discussed above to proceed.

At block 802, method 800 begins. At block 804, method 800 rehydrates a first culture medium and a first antibiotic with a bacterial suspension in a first antibiotic chamber to form a first mixture for an incubation period. The first medium and the first antibiotic may be a lyophilized mixture premixed into the first antibiotic chamber. The bacterial suspension can include a specific strain of bacteria to be analyzed (eg, to treat an infection caused by a bacterial strain in a patient). The first medium may include nutrients that allow bacteria to grow within the first antibiotic chamber while being killed by the first antibiotic.

At block 806, method 800 rehydrates a second culture medium and a second antibiotic with the bacterial suspension in a second antibiotic chamber to form a second mixture for the incubation period. The second medium and second antibiotic may be a lyophilized mixture premixed into the second antibiotic chamber. The bacterial suspension can include a specific strain of bacteria to be analyzed (eg, to treat an infection caused by a bacterial strain in a patient). The second medium may include nutrients that allow bacteria to grow within the second antibiotic chamber while being killed by the second antibiotic. In one example, the first antibiotic and the second antibiotic may be different antibiotics, or may include different doses of the same antibiotic.

At block 808, the method 800 confines the first mixture in the first filter chamber and the second mixture in the second filter chamber. After allowing the bacterial suspension to grow in the first and second antibiotic chambers, the mixture can be moved to the first and second filtration chambers.

At block 810, the method 800 induces a bacterial stress response by washing the first and second mixtures with a buffer solution to release a first metabolite in the first filtration chamber and a second metabolite in the second in the filter chamber. In the absence of culture medium, bacteria residing in the first and second filtration chambers can undergo a stress reaction. Stress reactions can cause bacteria to release metabolites.

In one example, promoters can be added to the bacteria. Accelerators increase the rate at which bacteria release metabolites. Metabolites can be measured to determine the amount of bacteria remaining in the first and second mixtures. The remaining amount of bacteria can determine the effectiveness of the first antibiotic and the second antibiotic.

At block 812, the method 800 transfers the first metabolite and the second metabolite over the plasmonic sensor for a Raman measurement. For example, the first filter chamber and the second filter chamber may include filters that allow the passage of metabolites while retaining bacteria. Metabolites can flow through the channel and span over the plasmonic sensor. Channels can be dried prior to Raman measurements. Raman measurements can determine the amount of metabolites in each channel. As described above, the measurement of metabolites can be used to determine the effectiveness of the first antibiotic and the second antibiotic.

At block 814, the method 800 generates a recommendation for the first antibiotic or the second antibiotic based on the Raman measurements. For example, a processor within the optical sensor can compare the Raman measurements within each channel. The channel with the lowest bacterial load determines the most effective antibiotic or the most effective antibiotic dose. The most effective dose of antibiotics can be recommended.

Although method 800 is illustrated with two passes, it should be noted that method 800 may be performed with more than two passes. Additionally, one of the channels can include a control group (eg, no antibiotics). At block 816, the method 800 ends.

It will be appreciated that variations of the above and other features and functions, or alternatives thereof, may be combined into other different systems or applications. Various substitutions, modifications, changes or improvements of which are not presently foreseen or contemplated may subsequently be made by those skilled in the art, which are also intended to be covered by the scope of the following claims.

100, 200, 500: plasmonic sensors; (microfluidic) wafers 102, 201, 202, 501: inlets 104 (104 ₁ , 104 ₂ ), 204 (204 ₁ to 204 _n ), 232,502 (502 ₁ to 502 _n ): channel 106 ₁ : (th a) antibiotic chamber 106 ₂ : (second) antibiotic chamber 108 ₁ , 108 ₂ , 208 , 508 (508 ₁ to 508 _n ): medium 110 ₁ : (first) filter chamber 110 ₂ : (second) filter chamber Chamber 112 ₁ , 112 ₂ , 212, 512 (512 ₁ to 512 _n ): filter 114, 214, 532: bacteria 116, 216, 534, 536: metabolites 120, 220, 520 (520 ₁ to 520 _n ), 720: plasmonic sensor 122 ₁ : (first) outlet ; outlet channel 122 ₂ : (second) outlet; outlet channel 206, 506 (506 ₁ to 506 _n ): antibiotic chamber 210, 510 (510 ₁ to 510 _n ): filtration chamber 213: lyophilized mixture; second lyophilized mixture 218 :毛細斷隙222:出口224:光學感測器226:光源230:通孔250:第一層252:第二層254:第三層256:基體258:光學透明層260:空氣400,600,800:方法402,404,406,408,410,412,414,416,418,602,604,606,608,610,612,802,804,806,808,810,812,814,816 : block 504 (504 ₁ to 504 _n ), 509 (509 ₁ to 509 _n ), 514 (514 ₁ to 514 _n ), 516 (516 ₁ to 516 _n ): valve 518 (518 ₁ to 518 _n ), 522 ( ₅₂₂₁ to _522n ): (outlet) channel 530: outlet 702, 704, 706: (microfluidic) channel

FIG. 1 illustrates a block diagram of an example microfluidic chip with plasmonic sensors of the present disclosure;

2 illustrates a top view of a block diagram of another example of a microfluidic chip with plasmonic sensors of the present disclosure;

3 illustrates a cross-sectional view of the example microfluidic wafer with plasmonic sensors illustrated in FIG. 2 of the present disclosure;

FIG. 4 illustrates an example operational flow diagram of performing an antibiotic susceptibility test (AST) using the plasmonic sensor illustrated in FIG. 2 of the present disclosure;

5 illustrates a top view of a block diagram of another example of a microfluidic chip with a plasmonic sensor of the present disclosure;

FIG. 6 illustrates a flow chart of an example operation of performing AST on a microfluidic wafer utilizing the plasmonic sensor illustrated in FIG. 5 of the present disclosure;

7 illustrates a top view of an example of how microfluidic channels may be routed over the plasmonic sensors of the present disclosure; and

8 illustrates an example flow diagram of a method of performing AST with a microfluidic wafer incorporating the plasmonic sensor of the present disclosure.

100: plasmonic sensor; (microfluidic) chip

102: Entrance

104 ₁ , 104 ₂ : channel

106 ₁ : (first) antibiotic chamber

106 ₂ : (Second) Antibiotic Chamber

108 ₁ , 108 ₂ : Medium

110 ₁ : (first) filter chamber

110 ₂ : (Second) Filtration Chamber

112 ₁ , 112 ₂ : filter

114: Bacteria

116: Metabolites

120: Plasmonic Sensor

122 ₁ : (first) exit; exit passage

122 ₂ : (second) exit; exit passage

Claims (15)

A microfluidic wafer comprising: a first channel including a first antibiotic chamber and a first filtration chamber, wherein the first antibiotic chamber includes a first culture medium and a first antibiotic rehydrated by a bacterial suspension, and the a first filtration chamber for filtration to separate bacteria from a first metabolite released by the bacteria in response to incubation with a buffer solution; a second channel comprising a second antibiotic chamber and a second filtration chamber, wherein the second antibiotic chamber comprises a second culture medium and a second antibiotic rehydrated from the bacterial suspension, and the a second filtration chamber for filtration to separate bacteria from a second metabolite released by the bacteria in response to incubation with the buffer solution; and a plasmonic sensor, wherein the first channel and the second channel pass over the plasmonic sensor, wherein the plasmonic sensor is used to measure the first metabolite and the second amount of metabolites. The microfluidic chip of claim 1, further comprising: a first inlet for providing the bacterial suspension; and A second inlet for providing the buffer solution. The microfluidic wafer of claim 1, wherein the first filter chamber and the second filter chamber each comprise a filter. The microfluidic chip of claim 1, further comprising: A multilayer fluid wafer comprising: a first layer comprising a channel to provide a fluid for controlling the flow of the bacterial suspension in the first channel and the second channel; A second layer comprising the first channel, the first antibiotic chamber, the first filter chamber, the second channel, the second antibiotic chamber, and the second filter chamber; a filter layer coupled to an opening of the first filter chamber and the second filter chamber; and A third layer including the plasmonic sensor and an optically transparent window over the plasmonic sensor. The microfluidic wafer of claim 4, further comprising: A capillary gap film layer between the first layer and the second layer. The microfluidic wafer of claim 1, wherein the first channel and the second channel are arranged in parallel on a common plane. The microfluidic wafer of claim 6, wherein the first filter chamber and the second filter chamber each include a first valve coupled with a first outlet channel and a first valve coupled with a second outlet channel Two valves, which include the plasmonic sensor. The microfluidic wafer of claim 1, wherein the first antibiotic chamber and the first filtration chamber comprise a single chamber. The microfluidic wafer of claim 1, wherein the first channel and the second channel are arranged in a serpentine shape to pass through the plasmonic sensor at different locations above the plasmonic sensor Second-rate. A microfluidic wafer comprising: an inlet for providing a bacterial suspension and a buffer solution; A plurality of channels, wherein each of the plurality of channels includes a respective medium chamber and a respective filtration chamber, wherein the plurality of channels include a different antibiotic and a respective medium chamber in the respective medium chamber. a medium in which the different antibiotics and the medium in the medium chambers are rehydrated by the bacterial suspension; a flow controller to move a mixture of the culture medium, the different antibiotics and the bacterial suspension to the respective filtration chambers, wherein the buffer solution is used to remove the culture medium from the mixture so that bacteria are released metabolites; and A plasmonic sensor to measure the amount of metabolites in the plurality of channels, wherein the metabolites are filtered from the mixture through a filter in the respective filter chamber. The microfluidic chip of claim 10, wherein one of the plurality of channels includes a control sample. The microfluidic wafer of claim 10, wherein the plasmonic sensor comprises a surface enhanced Raman spectroscopy (SERS) sensor. A method that includes: hydrating a first culture medium and a first antibiotic in a first antibiotic chamber with a bacterial suspension to form a first mixture for an incubation period; hydrating a second culture medium and a second antibiotic in a second antibiotic chamber with the bacterial suspension to form a second mixture for the incubation period; Confining the first mixture in a first filter chamber and the second mixture in a second filter chamber; Washing the first mixture and the second mixture with a buffer solution to induce an adverse reaction of bacteria to release a first metabolite in the second filter chamber and a second metabolite in the second filter chamber in the room; transferring the first metabolite and the second metabolite over a plasmonic sensor for a Raman measurement; and One of the recommendations for the first antibiotic or the second antibiotic is generated based on the Raman measurements. The method of claim 13, further comprising: Mixing an accelerator with the first mixture in the first filter chamber and the second mixture in the second filter chamber. The method of claim 14, wherein the promoter comprises a monophosphoribosyltransferase or a nucleoside monophosphate.

Images