Patent Application: US-201414503400-A

Abstract:
this disclosure describes portable bio - nano - chip assays of antiepileptic drugs using salivary samples . the bnc technology results in a more convenient , less expensive , and less time consuming sampling and analysis .

Description:
a disposable drug testing cartridge comprising a generally flat substrate having thereon individual bead sensors arranged in an array , wherein each bead sensor is a porous polymeric bead having a drug bound thereto , wherein said drug is selected from three or more of valproic acid , phenobarbital , phenytoin , clonazepam , carbamazepine , ethosuximide , felbamate , tiagabine , levetiracetam , lamotrigine , pregabalin , gabapentin , topomax , zonisamide , perampanel , lacosamide , topiramate , oxcarbazepine , and biological metabolites or derivatives of same . a disposable drug testing cartridge further comprising internal microfluidics on said substrate for carrying fluid to and from said bead sensors . a disposable drug testing cartridge further comprising a sample entry port . a disposable drug testing cartridge further comprising at least one reagent blister fluidly connected to said bead sensors . a disposable drug testing cartridge further comprising at least one waste fluid chamber fluidly connected to and downstream of said bead sensors . a disposable drug testing cartridge further comprising positive and negative control bead sensors and calibrator bead sensors having known amounts of a drug being calibrated . a disposable drug testing cartridge wherein every drug bead sensor is present in said array in at least duplicate . a disposable drug testing cartridge , wherein every drug bead sensor is present in said array in at least triplicate . a disposable drug testing cartridge wherein said drug is conjugated to said bead sensor via a linker . a disposable drug testing cartridge further comprising one or more of the following : one or more reagent chambers fluidly connected to and upstream of said array ; one or more waste fluid chambers fluidly connected to and downstream of said array ; a sample inlet upstream and fluidly connected to said one or more reagent chambers ; and wherein each bead sensor is a porous polymeric bead of size between 50 - 300 nm ± 10 %. an assay for the monitoring of anti - epilepsy drug concentration in saliva , said assay comprising : b ) immunologically testing said sample to determine the level of anti - epileptic drugs ; c ) wherein said testing is conducted on an array of agarose beads , conjugated to anti - epileptic drugs , and wherein signal from said array of agarose beads is analyzed by circular area of interest or line profiling or both . a assay of monitoring anti - epilepsy drug concentration in saliva , wherein said anti - epileptic drugs are selected from three or more of valproic acid , phenobarbital , phenytoin , clonazepam , carbamazepine , ethosuximide , felbamate , tiagabine , levetiracetam , lamotrigine , pregabalin , gabapentin , topomax , zonisamide , perampanel , lacosamide , topiramate , and oxcarbazepine and biological metabolites of same . a ) a microfluidic lab - on - chip based reverse competitive immunoassay that comprises a disposable cartridge and a separate reader , wherein said cartridge fits into a slot on said reader , and said reader performs said competitive immunoassay and outputs a result ; i ) a generally flat substrate having embedded microfluidic channels connecting an inlet port to an embedded downstream assay chamber having a transparent cover and containing a removable array of bead sensors ; ii ) one or more reagent chambers fluidly connected to and upstream of said assay chamber ; and iii ) one or more waste fluid chambers fluidly connected to and downstream of said assay chamber ; iv ) wherein each bead sensor is a porous polymeric bead of size between 50 - 300 nm ± 10 % having a drug conjugated thereto , wherein said drug is selected from three or more of valproic acid , phenobarbital , phenytoin , clonazepam , carbamazepine , ethosuximide , felbamate , tiagabine , levetiracetam , lamotrigine , pregabalin , gabapentin , topomax , zonisamide , perampanel , lacosamide , topiramate , and oxcarbazepine and biological metabolites of same ; and c ) wherein said reverse competitive immunoassay has a lower limit of detection for each of said drugs of & lt ; 50 ng / ml and a detection range of at least four orders of magnitude . a drug testing assay , said cartridge comprising 4 or more of said drugs . a kit , comprising the cartridge herein described wrapped in an airtight package , a vial of assay fluid , and an oral swab . the kit can include other components , e . g ., instructions for use . a ) a microfluidic lab - on - chip based reverse competitive immunoassay that comprises a disposable cartridge ; i ) a container closed with a removable cap ; ii ) a lower portion of said container having an assay fluid separated from an upper portion of said container by a piercable membrane ; iii ) said cap comprising a flexible bulb passing through said cap and fluidly connected to a hollow stem ending in a point , a lower portion of said stem being coated with an absorbent or bristled material for collecting a biological sample ; iv ) wherein said device is proportioned to store said cap with said stem and said swab in said upper portion of said container , but can reach said buffer when said point pierces said piercable membrane , and said flexible bulb can be used to draw up and deliver said buffer . i ) a generally flat substrate having embedded microfluidic channels connecting an inlet port to an embedded downstream assay chamber having a transparent cover and containing a removable array of bead sensors ; ii ) one or more reagent chambers fluidly connected to and upstream of said assay chamber ; iii ) one or more waste fluid chambers fluidly connected to and downstream of said assay chamber ; and iv ) wherein each bead sensor is a porous polymeric bead of size between 50 - 300 nm ± 10 % having a drug conjugated thereto , wherein said drug is selected from three or more of valproic acid , phenobarbital , phenytoin , clonazepam , carbamazepine , ethosuximide , felbamate , tiagabine , levetiracetam , lamotrigine , pregabalin , gabapentin , topomax , zonisamide , perampanel , lacosamide , topiramate , and oxcarbazepine and biological metabolites of same . the disclosure provides a biomarker assay cards for detecting anti - epileptic drugs in saliva and methods of analyzing the assay cards . specifically , bio - nano - chip ( pbnc ) biomarker assay cards are used to detect the concentration of aeds using non - invasive sampling of saliva . these cards can be ‘ read ’ using a lab - based system , a portable system , or a hand held device for home use . the lab - based system and a portable bnc reader system are seen in fig1 . both systems have a customized fluorescent microscopy equipped with a charged coupled device ( ccd ), a fluid delivery system , a computer and a stage for insertion of the bnc cell . generally , fluid is delivered in a microfluidic structure ( bnc chip ), flowing over and around the bead array , exiting underneath each microsphere through square fluid outlets . using this setup , known or unknown concentrations of analytes of interest are delivered to the bead array where they bind to the primary antibodies and are captured in a second step using fluorophore pre - labeled secondary antibodies . the fluorescent signal is measured using a fluorescent microscope . not shown is the hand held device . a hand held device is for home use to identify potentially hazardous dips in aed concentrations , much like a diabetic monitors blood sugar level . the hand held device will have the ccd and a micro - computer for displaying results , as well a temperature control if needed , fluidics for moving fluid , possibly reagents or reagent inputs . the ccd camera is only one option for assessing the light produced in the assay , and other photodetectors could be used including the common camera phone , cmos sensor , photo diode , and the like . further , the functionality can be provided in two components , e . g ., the hand held unit containing fluidics and / or pump , temperature control ( if needed ), and a separate camera phone with dedicated software application for reading and displaying results . the reader devices will be capable of analyzing the bnc assays for aed . fig2 displays the piping diagram of an exemplary pbnc chip like the ones used in the present disclosure . other footprints are also suitable and available . the present invention is exemplified with respect to fig4 - 10 . however , these figures are exemplary only , and the invention can be broadly applied to any aed capable of being monitored in saliva . the following examples are intended to be illustrative only , and not unduly limit the scope of the appended claims . in more detail , the bnc is a cartridge ( see e . g ., fig2 ) comprising a substrate having inlets and microfluidics for moving fluid and a plurality of individual bead sensors wherein each bead sensor is a porous polymeric bead having a competitor drug bound thereto ( either covalently bound or just absorbed , adsorbed , or adhered thereto ). additional beads for calibration and positive or negative control are also present . here the piping diagram shows the principle microfluidic channels and features of the p - bnc &# 39 ; s microfluidic network . the sample metering channel features ( i ) a port for sample input configured with a membrane filter , ( ii ) bubble trap , ( iii ) 100 μl capacity metered sample loop and ( iv ) a 20 μl sample overflow chamber leading to ( v ) an external vent . a passive pressure barrier ( vi ) prevents flow outside of the sample loop during loading . the right - hand fluid input port ( vii ) intersects the sample loop at a strategic location to evacuate the metered portion of sample toward the bead sensors . the lefthand fluid input hole ( viii ) is connected to the solid - state reagent storage chamber ( ix ) followed by an inline track - etch membrane filter ( x ) and bubble trap ( xi ). both the sample metering channel and reagent preparation channel confluence at the distal bubble trap , which forms a junction column with a single output . the common channel features six staggered herringbone mixer sets ( xii ) leading to the bead sensors ( xiii ) and a large capacity bilateral waste ( xiv ). the reservoir vents ( xv ) are covered with selectively permeable vent membranes for secure waste containment . the cartridge or card can also include blisters containing reagent fluids for use in said system . the reagents include wash buffers , reaction buffers , and the like , and can also include an anti - drug antibody coupled to a signaling reagent ( i . e . tracer ). the signaling reagent can be any reagent capable of providing a signal to the optical or energy sensing means , and preferably are fluorescent dyes , radioactive reagents , phosphorescent , chemi - luminescent or other energy emitting reagents . the reader devices can thus include mechanical actuators that apply pressure for the bursting of the blisters in a controlled fashion for the delivery of the said buffers and reagents . in other embodiments , the invention is the cartridge as described above , which can also include internal microfluidics on said substrate for carrying fluid to and from said bead sensors , as well as sample and / or fluid entry / exit port ( s ), together with a valve or access port , e . g ., a pinch valve or elastomeric stopper for accessing said internal microfluidics . in more detail , one embodiment of the invention disposable drug testing cartridge comprising a generally flat substrate having thereon individual bead sensors arranged in an array , wherein each bead sensor is a porous polymeric bead having a drug bound thereto , wherein said drug is two or more selected from a group containing : valproic acid , phenobarbital , phenytoin , clonazepam , carbamazepine , ethosuximide , felbamate , tiagabine , levetiracetam , lamotrigine , pregabalin , gabapentin , topomax , zonisamide , perampanel , lacosamide , topiramate and oxcarbazepine and biological metabolites of same . preferably , the drug testing cartridge has positive and negative control bead sensors and calibrator bead sensors , and every drug bead sensor is present in said array in at least duplicate or triplicate or more . we have exemplified the invention using spherical beads , but flat bead pads may also be used , as described in us20130130933 , incorporated herein by reference in its entirety for all purposes . of course , the image analysis may change based on the shape of the bead sensor . usually , the drug is conjugated to said bead sensor via a linker , but this can vary depending on the bead sensor chemistry . preferably , the bead sensor comprises crosslinked agarose , and the linker is a peptide or protein , such as bsa . fig4 shows the method of collecting oral fluid and how the results are displayed . oral fluid sample collected by a swab or pipette ( ai ) is extracted or diluted directly in assay buffer ( aii ) and then delivered to the microfluidic cell ( bi ) hosting bead sensors arrayed on a microchip ( bii ). step ( c ) shows the ccd captured images from a customized fluorescent microscope of a concentration - dependent response for a competitive - type p - bnc - based drug test ( i — control 0 , ii — 10 ng / ml of analyte and iii — 100 ng / ml of analyte ). noted are a ) the decrease in signal acquired on the drug sensors ( d sensor ) in response to the drug , b ) the absence of signal on negative control beads (− ye , ctl ) and c ) the consistency in signal intensity on the calibrator beads upon completion of the three independent assay runs . the schematic in c ( iv ) decodes the immune - components of this competitive assay approach . the current laboratory - based iteration of this p - bnc executes a ˜ 10 - minute test that reveals if there are aed drugs present in the body . this bead - based test functions with non - invasive oral fluid sampling , that involves use of an oral swab to brush the entire upper and lower gum line , and then insertion of the swab into a specimen collection tube to extract the sample into the assay fluid that includes the tracer antibody used in this competitive type of an immunoassay ( fig4 ). alternatively , the oral fluid is collected by pipette and diluted directly into the assay fluid . the sample / tracer mixture is then delivered to the p - bnc flow cell equipped with a bead - array platform . in the absence of drug in the sample , the tracer antibody specifically recognizes its corresponding drug sensor ( s ), producing a strong signal on the surface , as well as within the interior of the porous bead in the array . in the presence of drug in the sample the binding of the tracer is reduced in a drug specific , dose dependent manner . the array allows for 3 - 4 bead redundancy of bead sensors per drug target , which translates into higher accuracy and more precise measurements . as seen in the experiments , the bnc array has beads coated with bsa - drug conjugate , negative controls beads coated with bsa alone , and with calibrator beads used as internal controls . currently two elements are needed to measure the aed content in oral fluid . first is the lab - based imaging station with a fluidic control system ( seen in fig1 a ). this was utilized for all experiments discussed . however , a portable imaging system could also be used and is under development . further , there are also portable systems that are commercially available . the second element is the packaged microfluidic sample processing and immune - analysis bnc . this is the functional component for the detection and quantitation of the targeted drug and / or its metabolites . with these two elements , proof of concept experiments were conducted for phenytoin and phenobarbital . the aed test chips were developed and calibrated across a range of concentrations for these two target drugs . fig5 a is a proof of concept for 0 ng / ml and 1000 ng / ml phenytoin . fig5 b shows images of a proof of concept sequence of assay runs targeting 0 , 10 , 100 and 1000 ng / ml of a drug standard for phenytoin that ultimately allows for the generation of a dose response ( calibration ) curve , as shown in fig5 c , used to interpolate the concentration of the drug in oral fluid samples . fig5 d shows a dose response for phenobarbital for 0 , 1 , 10 , 100 , 1 , 000 and 10 , 000 ng / ml . fig5 e shows the dose response curve . the bead - based bnc competitive type of immunoassay was executed on the lab - on - a - chip ( loc ) system as follows : the tracer antibody was mixed with the saliva sample and the mixture delivered over a period of about 5 - 10 minutes ( 7 . 5 minutes ) to the array of beads in the microfluidic cell . the array had beads coated with bsa - drug conjugate , negative controls beads coated with bsa alone , and calibrator beads used as internal controls . fig6 displays the location of these beads within the bnc used . in the absence of drug in the sample , the tracer antibody efficiently recognized and bound to the bead sensors for which it was specific ( i . e . bead coated with the drug of interest ) and thus produced a fluorescent signal within and around the bead . in the presence of drug in the sample , which competed with the drug on the bead for binding to the tracer , the tracer - derived signal on the drug - sensitized bead was reduced in a dose dependent manner . fig5 a shows the results for phenytoin . as expected , the brightest signal was for the calibration beads and the sample beads for a phenytoin - free sample . upon introduction of a phenytoin - containing sample , the fluorescent signal decreased in intensity to barely being visible at a concentration of 1000 ng / ml , as shown in fig5 b . from the plot of concentration and signal intensity in fig5 c , the limit of detection ( lod ) was approximately 10 . 8 ng / ml . fig5 d - e shows the results for phenobarbital . here , the brightest signals were for the phenytoin sample beads and the phenobarbital sample beads for a phenobarbital - free sample . again , the fluorescence signal decreased in intensity as the concentration of phenobarbital increased . the concentration and signal intensity plot in fig5 e shows that phenobarbital had a high limit of detection at approximately 22 . 99 ng / ml . fig7 displays raw images of the bnc chips during the assay for a variety of control samples with known concentrations of phenobarbital and phenytoin . as the concentration of phy decreases , the beads in column 2 begin to appear around 10 ng of phenytoin . as the concentration of pbt increases , the beads in column 3 and 4 begin to decrease in signal intensity . based on these experiments , a plot of the concentration versus the linear profile intensity are shown in fig8 a - b . in fig8 a , it is indicated that the limit of detection based on this parameter was about 0 . 69 ng / ml for phy , and in fig8 b the limit of detection was about 18 ng / ml for pbt . the large difference in lod for both analytes was maintained . proof of concept was considered met by the above experiments because the bnc provided high signal to noise result , whereby the analyte - specific beads provided a significantly higher signal than the control beads in response to the tracer and the bnc demonstrated efficient competition between drug analyte and tracer , whereby the signal on drug - sensitized beads was significantly reduced in the presence of the specific drug . the presently disclosed multiplexed bnc assay chip was tested on a pilot population of volunteer epilepsy patients seen at the neurology practice of ut physicians at the texas medical center to determine the concentration of phenytoin ( fig3 b ) and phenobarbital ( fig3 c ) in these patients . as with most assay development , the success of the test relies heavily on the availability of high quality reagents . while most aeds can be analyzed , only two commonly used aeds were tested for the trial run because a plethora of commercial reagents exist for their assay , and these could be used as external controls . the patients were on a pbt and / or phy treatment regime . a serum sample and multiple saliva samples were obtained at each visit for each patient . oral fluid samples were collected by swabbing the entire upper and lower gum line with an oral swab which was then diluted by inserting the swab into a specimen collection tube containing aware messenger transfer matrix ( buffered sample ) or by collecting passive drool ( clear sample ) the buffered sample was immediately frozen at − 80 ° c . for analysis . the serum sample ( also called the ‘ gold standard ’) was processed by particle enhanced turbidimetric immunoassay ( petinia ) in a clinical chemistry laboratory . a control group of salivary samples was suspended in a phosphate buffer and assayed by gas chromatography - mass spectrometry ( gc / ms ) at a commercial toxicology laboratory . the saliva samples were run using sequentially diluted competitive mix against 3 clinical samples of pht and 5 of phb for a total of 8 samples . the results of this study are shown in fig9 . as can be seen in fig9 , the bnc easily detected the phb or pht in the buffered saliva . the estimated concentrations were favorable comparable to those seen in the gc / ms analysis . as such , the bnc provided reliable detection with high sensitivity . the assays benefited from automated image and data analysis macros developed specifically for this application . five dedicated image analysis “ probing ” strategies are shown in fig1 , including line profile ( lp ), circular area of interest ( caoi ), integrated density ( id ), circular profile ( cp ) and fixed aoi . the algorithm compiled results for each bead , statistical analysis with exclusion of outliers within each group of beads and output log files with the average , standard deviation and coefficient of variance for each group that can be inserted and further processed into a microsoft excel environment . intensity versus concentration calibration curves were constructed with best - fit regression analysis for determination of unknown sample concentration . data obtained from the testing of drug standards and zero antigen controls were then entered and processed to derive the dose response curves , as well as assay characteristics such as limit of detection , assay range and precision . the dose response data as well as data obtained from the testing of samples were entered into unknown prediction equations according to standard curves obtained for each analyte on the system to determine the drug concentrations . further enhancement in data quality was obtained by using image acquisition with various exposure times . the latter feature was developed with the flexibility that allows selective independent analysis for each assay using the optimal integration time for each target drug under the various conditions tested . line profile ( lp ) and circular area of interest ( caoi ) were the two image analysis methods that consistently provided the best results . hence , these two methods were selected and used extensively for the validation of the drug tests with respect to assay performance studies . for the line profile , a series of lines going through about 80 % of the beads were profiled for the maximum intensities ( or maxima ). because the signal is typically lower at the center of the beads , the product of a line profile is typically two maxima at the edge of the bead . all measurements were averaged and outliers identified and removed according to well established non - proprietary outlier removal routines ( median , grubb &# 39 ; s , or dixon tests ). for circular area of interest , a series of concentric areas centered on the center of the beads , and starting with a diameter of only a few pixels are drawn with increasing radii . for each of these circular areas , the average intensity per pixel was calculated and the circle was increased until it has exceeded the size of the bead by 10 %. the maximum signal obtained typically at the bead periphery can be determined from the highest circular area value . each of the following reference is incorporated by reference herein in its entirely . 61 / 498 , 761 , us20120322682 , wo2012154306 , wo2012065117 , wo2012065025 , wo2012021714 , wo2007134189 , wo2012065025 , 61 / 815 , 305 filed apr . 24 , 2013 . baumann rj . salivary monitoring of antiepileptic drugs . j . pharm . pract . 2007 ; 20 : 147 - 157 . greenaway c , ratnaraj n , sander j w , patsalos p n . saliva and serum lacosamide concentrations in patients with epilepsy . epilepsia 2011 ; 52 : 258 - 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