Patent Application: US-201615297776-A

Abstract:
in one embodiment , 3 - aminopropyltriethoxysilane functionalized graphene oxide wrapped sio 2 particle composite was prepared via the self - assembly process of aptes - go sheets and sio 2 particles . transmission electron microscopy and attenuated total reflectance - fourier transform infrared spectroscopy confirmed wrapping of the sio 2 particles by the aptes - go sheets . a biosensor based on electrochemical impedance spectroscopy was constructed and used to sensitively detect dengue dna and dengue rna via primer hybridization using different oligonucleotide sequences . the results demonstrated that the sio 2 @ aptes - go electrode material led to enhanced sensitivity , selectivity and detection limit , compared to both aptes - go and aptes - sio 2 . the three - dimensional structure , high surface area , electrical properties and the ability for rapid hybridization offered by the sio 2 @ aptes - go rendered this electrode material as ideal to use in the reported dengue impedimetric sensor .

Description:
for the purposes of promoting an understanding of the principles of the present disclosure , reference will now be made to the embodiments illustrated in the drawings , and specific language will be used to describe the same . it will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended . in response to the unmet need , a novel method and bio - sensing platform and biosensor are presented herein . we have developed a biosensor platform for the detection of the dengue virus and other mosquito borne viral diseases . the invention employs functionalized graphene oxide wrapped silica particles and impedance sensing to selectively detect dengue virus or other insect borne virus cdna and rna in solutions down to lam concentration . presently , arthropod - borne virus detection ( dengue , zika , chikungunya and west nile virus etc .) rely on enzyme - linked immune - adsorbent assays ( elisa ) and sensitive polymerase chain reaction ( pcr ) testing . these tests require complex and time consuming sample preparation , sample transport to specialized laboratories with trained personnel , and are also susceptible to false positive results . there are currently no point of care devices for the detection of the zika virus ( or any other arthropod - borne viral disease ) offering in - situ , rapid , ultra - sensitive , accurate sensing capabilities for both personal and clinical use . this disclosure is to provide a biosensing technology that can be readily implemented for various insect borne virus detection , including various dangerous viruses such as zika virus , west nile virus etc . the biosensor detection can be integrated into inexpensive platforms for personal and clinical point of care applications . equipping the device with easy to interpret output monitoring and wi - fi data sending capabilities , both the end user and the health care team can instantaneously respond to the test results . biosensors are bioanalytical tools that measure the presence of analytes by combining the sensitivity of biomolecular recognition elements with a physical transduction mechanism . they play a major role in the development of time - effective , low - cost and easy - to - use analytical tools and are particularly suitable for miniaturization and portability . their advantages include their high sensitivity and specificity provided by the biocatalytic or biorecognition sensing elements . various kinds of biosensors ( enzyme - based , immunosensors , dna - sensors ) have been broadly studied but only few of them have been successfully commercialized . the global biosensors market is expected to grow from $ 6 . 72 billion in 2009 to $ 22 . 5 billion in 2020 . most of the developed biosensors address medical needs and are used for diagnostics purposes . applications in environmental and agricultural fields , and particularly for anti - terrorist activity and homeland security , are also rapidly increasing . for example , optical biosensors have now the highest sensitivity , approaching theoretical limits of interface sensitivity , which is critical for detection of drug candidates , viruses , or pathogens . electrochemical biosensors function on the basis of correlating the electronic signal given off upon interaction of the biological recognition element with the analyte . there are different types of electrochemical biosensors , which measure the electrical properties of an electrode surface and the binding kinetics of molecules . in particular , electrochemical impedance spectroscopy ( eis ) can measure the changes of the electrical properties of a surface arising from the interaction with the captured analyte , while minimizing sample damage during measurements . disclosed herein is a method of detecting , monitoring , and ultimately controlling the occurrence of insect - borne viral diseases . these viral diseases can include but are not limited to dengue , yellow fever , chikungunya , west nile diseases and zika fever . also disclosed herein is a biosensor platform and biosensor system for accomplishing this detection and monitoring of insect - borne viral diseases . the method for detecting and monitoring insect - borne diseases , includes placing a biosensor in a predetermined location . this predetermined location can be chosen , as an example , by a user based on the presence of insects that are vectors for disease transmission . a sample of nucleic acid ( na ) is obtained from such insects . the sample of na is placed on a sensing device , and the sensing device is configured to measure a change in resistance to an applied electrical current when the na sample corresponds to the virus - causing disease . the sample of na ( dna or rna ) is probed and utilized in the sensing device to obtain impedance and resistance data . the impedance and resistance data can be used to determine if a particular virus is present in the insect . these results can be transmitted to the user , and the user ( for example a disease control center or medical monitoring facility ) can then utilize the data to take preventative measures such as selective fumigation . such measures can be rapidly implemented to prevent further occurrences of such diseases by destroying the infected viral disease vectors . there are different methods for obtaining the na sample . for example , the sample of na can be obtained from a person &# 39 ; s blood sample , the individual having already been bitten by the insect . the sample of na from the insect can also be obtained by enticing the insect to bite a membrane . the membrane can be configured to extract the virus sample and therefore its rna from the insect through conventional microfluidics devices . also disclosed herein is a biosensor platform . the biosensor platform includes an electrode material configured to be coupled to at least one functionalized particle . the platform can host functionalized particles for detecting different viral rna . the biosensor platform can also feature the electrode material disposed on the screen printed supporting strip . in one embodiment , the supporting strip can be a rigid substrate . the functionalized material consists of positive functional groups on the surface of the material . the material can be an electrically conducting material . this electrically a conducting material can be graphene . the electrode material can be conductive material such as platinum , copper etc . the functionalized material can be silicon dioxide . alternative materials that can be used are conductive materials that have high surface areas , such as for example conducting polymers such as poly ( 3 , 4 - ethylenedioxythiophene ) ( pedot ) or poly ( p - phenylene sulfide ( pps ), or functionalized metallic nanoparticles ( e . g . silver ), or silver nanoparticle loaded graphene . in another embodiment , the biosensor can be implemented into a biosensor system , which has at least one functionalized material and a nanoparticle - based amperometric biosensor . the nanoparticle - based amperometric biosensor can be configured for nucleic acid ( na ) purification and extraction from a grinded insect sample and subsequent probing of the na to be compared to the genome of the disease causing virus . a wireless data transmission device can be coupled to the biosensor system , and further coupled to a power source . in yet another embodiment , at least one probe is coupled to the biosensor . these probes can be configured to allow detection of a particular virus . the biosensor system can also have a global positioning system ( gps ). the gps can be coupled to the wireless data transmission device to permit transmission of location data . the power source can be a battery . the power source in yet another embodiment can be configured to be self - sustaining , for example , it can be configured to run on solar power , and thereby permit unmanned and remote operation . silica particles were synthesized by the modified stober method ( lei et al ., 2014 ): 9 . 01 ml of di water , 50 ml of ethanol ( 100 %, koptec ) and 1 . 37 ml of ammonium hydroxide ( nh 3 28 ˜ 30 %, sigma - aldrich ) were mixed together and 3 . 2 ml of tetraethyl orthosilicate ( teos , 99 %, fluka ) was added drop - wise into the mixed solution . after 1 hour , the synthesized particles were separated from the mixed solution using an ultracentrifuge ( eppendorf ag 22331 , hamburg , germany ) spinning at 14 . 5 krpm , and then repeatedly washed using ethanol at least six times . the washed particles were first dried at 354k for 6 hr and then were grinded into fine particles . subsequently , they were heat - treated in air at 383k for 24 hr . the end product was finally grinded again . the graphene oxide ( go ) sheets were prepared through chemical oxidation of graphite particles by a modified hummer &# 39 ; s method ( hummers and offeman , 1958 ). graphite powder , 0 . 85 gr , ( 99 . 9995 %, alfa aesar ) and 23 ml of h 2 so 4 ( 95 ˜ 98 %, sigma - aldrich ) were stirred for 8 hrs . next , 3 . 0 g of kmno 4 (≧ 99 %, sigma - aldrich ) was slowly added at a temperature below 294k . the mixture was then heated at 314k while constantly stirring it for thirty minutes , and subsequently for an additional 45 minutes at 344 k . the solution was next diluted with 46 ml of deionized water ( di ) and heated at 373k for 30 min . the oxidation reaction was terminated by adding 140 ml of di water together with 10 ml of h 2 o 2 solution ( 30 %, macron ) after being cooled down to room temperature . the oxidized graphite particles were washed and filtered several times using a 10 % hcl ( 37 %, sigma - aldrich ) solution with di water , then dried at 333 k under vacuum . exfoliation was conducted to ultimately synthesize go sheets by using a bath sonicator ( cole - parmer 8891 ; cole - parmer , vernon hills , ill ., usa ) and a branson digital sonifier 102c ( branson , danbury , conn ., usa ). positively charged graphene oxide was prepared using 3 - aminopropyltriethoxysilane ( aptes , 99 %, sigma - aldrich ) by the reflux method . 20 mg of go was first dispersed in 100 ml of toluene ( 99 . 8 %, sigma - aldrich ). the go dispersed solution was degassed using nitrogen gas ( 99 . 995 %) for 15 min to remove oxygen within the solution , then 0 . 6 ml of aptes was injected into the mixed solution . the solution was stirred for 3 hr at 303 k in a nitrogen atmosphere and then refluxed at 383 k for 10 hr under an inert nitrogen gas environment . the aptes grafted - go ( aptes - go ) was rinsed several times with toluene , ethanol and di water , using an ultracentrifuge . each material was dispersed in aqueous solution using 20 mg of aptes - go , 4 mg of sio 2 particles and di water separately . the aptes - go solution was dropped into the sio 2 dispersed solution under ultra - sonication , then stored for 24 hr . the coagulated sio 2 @ aptes - go composite was rinsed using an ultracentrifuge with di water several times . an oligonucleotide primer was designed and immobilized on the material as a probe for dna hybridization and its sequence is 5 ′- ggt - tgg - atg - cgc - gca - tct - att - ctg - acc - cac - tgg - 3 ′ ( seq id no : 1 ). example 2 : a functionalized graphene based 3 - dimensional structured material for rna detection silica particles were prepared by the modified stober method in the same manner as in synthesis example 1 , the graphene oxide ( go ) sheets were prepared in the same manner as in synthesis example 1 . the sio 2 @ aptes - go composite was prepared in the same manner as in synthesis example 1 . an oligonucleotide primer was designed and immobilized on the material as a probe for rna hybridization and its sequence is 5 ′- ata - caa - tgt - ggc - atg - tca - cac - gtg - gcg - 3 ′ ( seq id no : 2 ). the complementary rna was extracted from dengue virus infected mosquito cell lines . c6 / 36 cells infected with dengue virus strain 16681 at an moi of 2 after 70 - 80 % of confluence cell growing . the infected cells were washed using 1 × pbs solution and trizol extraction was performed using 1 ml of trizol ls reagent . a volume of 0 . 25 ml chloroform was added to the homogenized sample , then incubated at temperatures ranging from 288 to 303k for 5 minutes . the solution was incubated again for 10 minutes at the same temperature after vigorous shaking for 15 seconds . after incubation , the sample was centrifuged at 12 , 000 × g for 15 min . at a temperature of 277k . rna was precipitated using isopropanol after collecting the upper aqueous phase and incubated for 10 min on ice , thereafter , it was centrifuged at 12 , 000 × g for 10 min . the precipitates were collected and washed using 75 % ethanol and 7500 × g centrifugation at 277k for 5 minutes . the washed precipitates finally resolved in rnase free water after drying . silica particles were prepared by the modified stober method in the same manner as in synthesis example 1 , aptes - grafted sio 2 particles were prepared through the reflux method , similarly to the preparation of aptes - go . an amount of 0 . 1 g of sio 2 particles and 150 ml of ethanol were mixed together using ultra - sonication for 30 min , and then the solution was degassed using nitrogen gas . a volume of 0 . 5 ml of aptes was injected into the degassed solution under nitrogen gas atmosphere and the mixture solution was refluxed at 353 k for 6 hrs . afterwards , the functionalized sio 2 was washed several times with ethanol and di water using an ultracentrifuge . an oligonucleotide primer was designed and immobilized on the material as a probe for dna hybridization and its sequence is 5 ′- ggt - tgg - atg - cgc - gca - tct - att - ctg - acc - cac - tgg - 3 ′ ( seq id no : 1 ). silica particles were prepared by the modified stober method in the same manner as in synthesis example 1 . positively charged silica particles were prepared in the same manner as in synthesis example 2 . an oligonucleotide primer was designed and immobilized on the material as a probe for rna hybridization and its sequence is 5 ′- ata - caa - tgt - ggc - atg - tca - cac - gtg - gcg - 3 ′ ( seq id no : 2 ). the complementary rna was extracted from dengue virus infected mosquito cell lines in the same manner as in synthesis example 2 . silica particles were prepared by the modified stober method in the same manner as in synthesis example 1 . positively charged silica particles were prepared in the same manner as in synthesis example 2 . an oligonucleotide primer was designed and immobilized on the material as a probe for rna hybridization and its sequence is aac - ctt - cgc - tct - att - ctc - atc - agt - ttc - atg ( seq id no : 3 ) comparative example 1 : a functionalized graphene based 2 - dimensional sheet for dna detection the graphene oxide ( go ) sheets were prepared in the same manner as in synthesis example 1 . an oligonucleotide primer was designed and immobilized on the material as a probe for dna hybridization and its sequence is 5 ′- ggt - tgg - atg - cgc - gca - tct - att - ctg - acc - cac - tgg - 3 ′ ( seq id no : 1 ). the surface charge was measured using a malvern zetasizer ( nano z , malvern , uk ). we employed 0 . 02 wt % of material dispersed di water solution to check zeta ( ζ ) potential . the smoluchowski model was used in order to convert from the electrophoretic mobility to ζ potential . all the functionalized materials showed positive charge due to the presence of amine groups on the surface of the materials ( fig1 ). the average values were + 24 . 4 mv for the material of comparative example 1 , − 34 . 7 mv for the materials of example 3 and example 4 , and + 16 . 7 mv for the materials of example 1 and example 2 . the structure of sio2 particle and sio 2 @ aptes - go composite particle were observed using a fei - tecnai transmission electron microscope ( tem ). fig2 ( a ) and ( b ) clearly shows the microstructure of sio 2 particles and the functionalized graphene based 3 - dimensional structured material . the average size of sio 2 was 206 nm ( fig2 a ) and the fig2 b shows the wrapped structure by the positively charged graphene oxide sheets on the sio2 particle . a 5 mm platinum electrode was first cleaned by polishing with alumina paste and washing by sonication with di and ethanol solution , and subsequently used for biosensor platform fabrication . a concentration of 0 . 2 wt . % of the positively functionalized material ( aptes - sio 2 , aptes - go , and sio 2 @ aptes - go ) solution was prepared using di water . a volume of 20 ul of the mixture was dropped on the pt electrode , and then residual materials were removed by washing with di water after 10 min . the primer was immobilized on the positively functionalized material layer at room temperature ; excessive primers that were not successfully immobilized were removed after primer immobilization , 40 minutes for dna target and 2 hrs for rna target separately , through rinsing with di water . the electrodes were incubated with various concentrations ( 10 pm , 1 fm , and 1 am ) of complementary dna and 10 pm non - complementary dna in a 10 mm pbs solution at 333 k for 5 hrs . finally , the electrodes were washed with di water to remove unhybridized dna . rna hybridization was separately conducted under same condition with that of dna hybridization under the various concentration of rna in rnase free water . electrochemical impedance spectroscopy ( eis ) was performed in 10 mm pbs containing 10 mm k4 [ fe ( cn ) 6 ] 4 − / k3 [ fe ( cn ) 6 ] 3 − electrolyte using a bio - logic potentiostat ( sp - 150 , bio - logic sas , france ). a three - electrode electrochemical cell for eis analysis was prepared with the as - fabricated biosensor electrode as a working electrode , a pt wire counter electrode , and an ag / agcl reference electrode . impedance spectra were recorded in the frequency range of 100 mhz to 100 khz , with 10 mv amplitude . using the impedance data , the charge - transfer resistance ( r ct - layer ) of aptes - sio 2 , aptes - go , and sio 2 @ aptes - go , respectively , of a immobilized primer layer ( r ct - primer ), and of a hybridized complementary dna or rna layer ( r ct - com ) were analyzed using the randles &# 39 ; model ( rushworth and hirst , 2013 ) and calculated from subtracting r ct the probe immobilized electrode , from the rct after incubation using test solution . referring to fig5 and 7 , it is confirmed that all of the particles with positive charge were able to detect complementary target with these values . fig3 is showing the charge transfer resistance change from the hybridization of dna on the probe immobilized surface , r ct - com . the r ct - com values for aptes - go ( comparative example 1 ), aptes - sio 2 ( example 3 ), and sio2 @ aptes - go ( example 1 ) are 6 . 37 ± 1 . 97 ω , 22 . 22 ± 1 . 7ω , and 33 . 29 ± 1 . 24ω , respectively . fig4 represents that the material of example 1 ( sio2 @ aptes - go ) is able to detect down to latto - molar concentration of dengue serotype 2 dna . fig5 shows the results in terms of after 10 pm and 1 am rna hybridization of aptes - sio 2 and sio 2 @ aptes - go . the r ct - com values were 30 . 19 ± 4 . 02ω for aptes - sio 2 ( example 4 ), and 53 . 72 ± 4 . 82ω for sio 2 @ aptes - go ( example 2 ), respectively . all the r ct - com values were from the half circle diameter increasing after target hybridization ( fig6 ). fig6 represent 1 fm dengue dna detection result . very recent work showed successful detection of 1 pm zika rna by using the same principles of impedance sensing and the same graphene - oxide based electrode materials ( fig7 ). oligonucleotide probes complementary to zikv rna with the sequence aaccttcgctctattctcatcagtttcatg ( seq id no : 3 ) have been prepared and used to detect zika virus of 1 pm concentration . this initial result serves as proof of concept for the biosensing platform feasibility and transferability to zika detection . the probe immobilized functional particle detected 1 pm zika rna ( fig7 ) upon incubation of several hours of the probe with zika rna . we have employed electrochemical impedance spectroscopy ( eis ) to demonstrate the working principles of our developed virus bio - sensing technology utilizing commercial laboratory equipment . eis is a versatile tool that measures the electrical impedance of a system as a function of frequency . it is a versatile technique widely employed in diverse fields such as electrochemistry , medicine , biology , food science , geology , etc . however , conventional eis methods , in particular those performed in laboratory instruments are very slow at low frequencies , a frequency range often characteristic of the response of biosensors . thus , an extensive review of the available methods and electronic devices that can perform the key processes pertaining the biosensor platform is required . these parameters include analyte recognition , signal transduction , readout and data transmission . components and measuring techniques will be selected with a focus on specificity , speed , portability , and low costs . in addition , discrete component will be selected to optimize the detection sensitivity of the zika biosensor and assembled in a circuit breadboard together with the readout and data transmission electronics ; intrinsic noise and environmental interference cancellation will be considered . point of care bio - 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