Patent Application: US-201113279083-A

Abstract:
microfluidics has made great progress in integrating many aspects of biological analysis and testing into the microscale . one aspect which has proven challenging to miniaturize has been fluorescence testing , as a complete fluorescence system requires an integrated light source , detector and filters to filter out the excitation light from the detector . here we demonstrate that with polarization filtering of the excitation light and multiple dye sources modulated at different frequencies , a high - sensitivity , multi - dye system with one detector can be realized . simultaneous detection and quantition of a mixture of two different dyes is demonstrated with no physical change in the measurement setup . the degree of interaction of the dyes is measured . this system is readily adaptable to integrated lab - on - a - chip microfluorescence .

Description:
for proof - of concept , two different fluorescent dyes having different excitation wavelengths , as shown in fig2 , were selected . to use this method , it is necessary for the two dyes to have distinct excitation wavelengths . as will be discussed , it is the frequency of the response , and not the color , that a locked - in photodetector detects ; the two dyes could overlap in emission wavelength and still be distinguished by lock - in detection . the dyes selected are rhodamine 6g ( rh6g ), which has an absorption maximum at 530 nm and a peak emission wavelength of 566 nm ; and trisodium 8 - methoxypyrene - 1 , 3 , 6 - trisulfonate ( pts1 ) which has its absorption maximum at 404 nm and a peak emission wavelength of 430 nm . both the emission and excitation wavelengths of the two dyes are distinct . fig3 shows the emission spectra of the two leds used as excitation sources . as can been seen , the green led has an emission peak of 525 nm and extends about 100 nm , and the blue / uv has an emission peak of 400 nm and extends about 50 nm . there is no significant overlap in emission spectra . to make precise concentrations of the two dyes , precise amounts of dye powder were weighed on a microbalance and mixed with specific volumes of distilled water . 1 mm standard solutions were made with both dyes , and then further diluted as standards to make more dilute solutions . every solution is stirred thoroughly to ensure uniform concentrations . concentrations from 10 − 3 to 10 − 9 m were mixed to create a full experimental matrix of mixtures of both dyes at all ranges of concentration in a factor - of - ten dilution series . the schematic diagram of the setup is shown in fig4 . two leds are driven separately with different frequencies , and two polarizing films ( edmund optics nt45 - 667 ) oriented at 90 degrees are used to isolate the excitation light from the emission light . a 3 ml cuvette holder between them contained the dye sample . in this experiment , the cuvette is a standard square cuvette with an optical path length of 10 mm and capacity of 3 . 5 ml . before determining the modulation frequencies of the separate diodes , the modulation frequency of the system was evaluated to ensure that the diode response is not limited by the pin diode or other electronics . an led was driven directly by a signal generator by a sine wave from 0 to ˜ 2 v at frequencies from 1 khz to 100 khz and the ac response at the output of the transimpedance amplifier measured by an ac voltmeter . the 3 db bandwidth of system is shown in fig5 . the bandwidth of the system was 63 khz another way of evaluating system speed is in the time response to a square wave input . the led was pulsed with a square voltage , and the output out of the transimpedance amplifier was measured on the scope . this system time response is shown in fig5 , which has rise time ( signal rise time 10 % to 90 % of its maximum value ) of 5 ms or ˜ 20 khz . the modulation frequencies for the leds were chosen to be 2 and 3 khz , well below either cutoff . the input voltage ranges on the led are picked so as to not saturate the detector when both leds are on and give clean signals for both 2 khz and 3 khz signal . the crossed polarizers reduce the magnitude of the detector photocurrent due to excitation light by about 25 db . the signal output from detector goes through transimpedance amplifier to translate the nanoamperes of photocurrent into tenths of volts of response . dye samples for both rh6g by itself , and pts1 by itself are made with concentrations from 0 . 1 mm to 0 . 01 nm . the dc photovoltage response directly out of the photodetector is measured and plotted in fig6 . the horizontal line in both figures demonstrates the response for a 0 m concentration of distilled water . this gives a background signal floor to the fluorescence measurements . the minimum concentration that can be read , the limit of detection , is determined by the amount of noise in the signal compared to the amplitude of the signal . the noise level of the signal is estimated by taking the variation of the time - series measurements of the background water . at the lowest concentration measured of 10 nm for pts1 and 100 pm for rh6g , the signal was 10 mv higher than the background , with a signal - to - noise ratio of ˜ 3 . the dc response demonstrated detectability down to about the 10 nm range . however , measurement of dc photocurrent means the system is susceptible to ambient background light variations . in addition , without wavelength - specific optical filters , the system cannot distinguish between different colors of emission . both of these disadvantages are overcome by using lock - in detection . with lock - in detection , the leds are driven at a specific frequency , and the photodetector is connected to a transimpedance amplifier . the output of the transimpedance amplifier is connected to a princeton electronics digital lock - in amplifier , locked to the source frequency . the response measured by the lock - in is the response only at the frequency of the driven led , and noise at other frequencies is not detected . such lock - in detectors have excellent ability to discriminate between signal and noise , and can detect nanovolts of signal in the presence of noise many orders of magnitude larger . use of the lock - in eliminated dc ambient noise , and allowed emission light from different dyes to be distinguished by modulation frequency . the emission light at 2 khz reflected the concentration of dye excited by a 2 khz source , largely regardless of the background amount of dye driven by a 3 khz led . this technique also eliminated cross - fluorescence ( in which luminescence form one dye acted as an excitation for another ); the cross - fluorescence signal will be at a different frequency , and so ignored by the lock - in . a complete experimental matrix with mixtures of all possible concentrations ( in steps of factors of 10 ) of pts1 and rh6g in the range of 1 nm to 0 . 1 mm was constructed . for each mixture , the lock - in response at 2 and 3 khz was measured and plotted against dye concentration . the results are shown in fig7 a and 7b . each line represents a response with a particular background concentration of the other dye . this indicates the crosstalk between the dyes , or amount by which the background concentration of pts1 , for example , affects the measurement of the concentration of rh6g . the lock - in measurements of the rh6g dye response shows a slight dc level shift due to pts1 concentration . as the concentration of pts1 increases , the signal increases slightly . the pts1 photo response curves for different level of rh6g are similar with slight dc level shift . this is believed to be largely because of excitation light scattering by the other dye , which reduces the effective polarization filtering . because the response is inherently non - linear , very small levels of additional response can lead to significant shift in the overall curve and change in apparent concentration . based on these results , it is believed that , with prior calibration and appropriate algorithms for mathematical fitting , with measurement of the response at both 2 and 3 khz , the concentrations of rh6g and pts1 can be accurately determined . further , this technique may be extended to different dyes or mixtures of more than two dyes . this technique , which requires no color - dependent filters but distinguishes emission of two different dyes by color , can be easily extended to thin , lab - on - chip systems . two integrated organic light sources combined with single organic photo detector and integrated polarizers would realize this system in a small , flat , disposable configuration . see , e . g ., [ 7 , 8 ]. the system demonstrated good discrimination between two dyes to very low levels and detectability of 10 nm . the present system and method may be used on a micromachined flow cytometry system , to classify fluorescently tagged cells as they pass through a small volume flow passage . likewise , the system can be parallelized , to concurrently analyze a plurality of flow passages . in some cases , single illumination sources can be used for multiple flow passages . the flow cytometry system may be used , for example , to detect tumor cells . see , e . g ., www . ivdiagnostics . com / about . html , www . purdue . edu / uns / x / 2007b / 070904lowpnas . html , 2007 national academy of sciences paper : www . ncbi . nlm . nih . gov / pmc / articles / pmc1913863 /, www . electroiq . com / index / display / nanotech - article - display / 4553408772 / articles / small - times / nanotechmems / mems / microfluidics / 2010 / september / lab - on - chip - project . html , each of which is expressly incorporated herein by reference . various aspect of the technology may be implemented on an automated computer using known components . the computer may be controlled in accordance with a tangible computer - readable medium , such as a magnetic disk , optical disk , flash memory , and other physical systems . the automated computer itself typically comprises a processor , which may be cisc , risc , simd , or other types . embodiments hereof may be implemented as a method , apparatus , or article of manufacture using standard programming and / or engineering techniques to produce software , firmware , hardware , or any combination thereof to control a computer to implement the disclosed embodiments . the term “ article of manufacture ” ( or alternatively , “ computer program product ”) encompasses a computer program accessible from any computer - readable device , carrier , or media . for example , computer readable media can include but are not limited to magnetic storage devices ( e . g ., hard disk , floppy disk , magnetic strips , etc . ), optical disks ( e . g ., compact disk ( cd ), digital versatile disk ( dvd ), blueray disks , etc . ), smart cards , and flash memory devices ( e . g ., card , stick ). additionally it should be appreciated that various types of information can be communicated using a carrier wave such as those used in transmitting and receiving electronic mail or in accessing a network such as the internet or a local area network ( lan ). in some cases , these carrier waves reside within tangible media , and can be deemed non - transitory . of course , those skilled in the art will recognize many modifications may be made to this configuration without departing from the scope or spirit of the disclosed embodiments . referring now to fig8 , there is illustrated a block diagram of a computer operable to execute the disclosed architecture . in order to provide additional context for various aspects disclosed herein , fig8 and the following discussion are intended to provide a brief , general description of a suitable computing environment 1100 in which the various aspects can be implemented . while the one or more embodiments have been described above in the general context of computer - executable instructions that may run on one or more computers , those skilled in the art will recognize that the various embodiments also can be implemented in combination with other program modules and / or as a combination of hardware and software . generally , program modules include routines , programs , components , data structures , etc ., that perform particular tasks or implement particular abstract data types . moreover , those skilled in the art will appreciate that the inventive methods can be practiced with other computer system configurations , including single - processor or multiprocessor computer systems , minicomputers , mainframe computers , as well as personal computers , hand - held computing devices , microprocessor - based or programmable consumer electronics , and the like , each of which can be operatively coupled to one or more associated devices . the illustrated aspects may also be practiced in distributed computing environments where certain tasks are performed by remote processing devices that are linked through a communications network . in a distributed computing environment , program modules can be located in both local and remote memory storage devices . a computer typically includes a variety of computer - readable media . computer - readable media can be any available media that can be accessed by the computer and includes both volatile and nonvolatile media , removable and non - removable media . by way of example , and not limitation , computer - readable media can comprise computer storage media and communication media . computer storage media includes both volatile and nonvolatile , removable and non - removable media implemented in any method or technology for storage of information such as computer - readable instructions , data structures , program modules or other data . computer storage media includes , but is not limited to , ram , rom , eeprom , flash memory or other memory technology , cd - rom , digital video disk ( dvd ) or other optical disk storage , magnetic cassettes , magnetic tape , magnetic disk storage or other magnetic storage devices , or any other medium which can be used to store the desired information and which can be accessed by the computer . communication media typically embodies computer - readable instructions , data structures , program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism , and includes any information delivery media . the term “ modulated data signal ” means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal . by way of example , and not limitation , communication media includes wired media such as a wired network or direct - wired connection , and wireless media such as acoustic , rf , infrared and other wireless media . combinations of the any of the above should also be included within the scope of computer - readable media . with reference again to fig8 , the exemplary environment 1100 for implementing various aspects includes a computer 1102 , the computer 1102 including a processing unit 1104 , a system memory 1106 and a system bus 1108 . the system bus 1108 couples system components including , but not limited to , the system memory 1106 to the processing unit 1104 . the processing unit 1104 can be any of various commercially available processors . dual microprocessors and other multi - processor architectures may also be employed as the processing unit 1104 . the system bus 1108 can be any of several types of bus structure that may further interconnect to a memory bus ( with or without a memory controller ), a peripheral bus , and a local bus using any of a variety of commercially available bus architectures . the system memory 1106 includes read - only memory ( rom ) 1110 and random access memory ( ram ) 1112 . a basic input / output system ( bios ) is stored in a non - volatile memory 1110 such as rom , eprom , eeprom , which bios contains the basic routines that help to transfer information between elements within the computer 1102 , such as during start - up . the ram 1112 can also include a high - speed ram such as static ram for caching data . the computer 1102 further includes an internal hard disk drive ( hdd ) 1114 ( e . g ., eide , sata ), which internal hard disk drive 1114 may also be configured for external use in a suitable chassis ( not shown ), a magnetic floppy disk drive ( fdd ) 1116 , ( e . g ., to read from or write to a removable diskette 1118 ) and an optical disk drive 1120 , ( e . g ., reading a cd - rom disk 1122 or , to read from or write to other high capacity optical media such as the dvd ). the hard disk drive 1114 , magnetic disk drive 1116 and optical disk drive 1120 can be connected to the system bus 1108 by a hard disk drive interface 1124 , a magnetic disk drive interface 1126 and an optical drive interface 1128 , respectively . the interface 1124 for external drive implementations includes at least one or both of universal serial bus ( usb ) and ieee 1394 interface technologies . other external drive connection technologies are within contemplation of the one or more embodiments . the drives and their associated computer - readable media provide nonvolatile storage of data , data structures , computer - executable instructions , and so forth . for the computer 1102 , the drives and media accommodate the storage of any data in a suitable digital format . although the description of computer - readable media above refers to a hdd , a removable magnetic diskette , and a removable optical media such as a cd or dvd , it should be appreciated by those skilled in the art that other types of media which are readable by a computer , such as magnetic hard drives , flash memory cards , optically readable media , and the like , may also be used in the exemplary operating environment , and further , that any such media may contain non - transitory computer - executable instructions for performing the methods disclosed herein . a number of program modules can be stored in the drives and ram 1112 , including an operating system 1130 , one or more application programs 1132 , other program modules 1134 and program data 1136 . all or portions of the operating system , applications , modules , and / or data can also be cached in the ram 1112 . it is appreciated that the various embodiments can be implemented with various available operating systems or combinations of operating systems . a user can enter commands and information into the computer 1102 through one or more wired / wireless input devices , e . g ., a keyboard 1138 and a pointing device , such as a mouse 1140 . other input devices ( not shown ) may include a microphone , a remote control , a joystick , a game pad , a stylus pen , touch screen , or the like . these and other input devices are often connected to the processing unit 1104 through an input device interface 1142 that is coupled to the system bus 1108 , but can be connected by other interfaces , such as a parallel port , an ieee 1394 serial port , a game port , a usb port , an interface , etc . a monitor 1144 or other type of display device is also connected to the system bus 1108 through an interface , such as a video adapter 1146 . in addition to the monitor 1144 , a computer typically includes other peripheral output devices ( not shown ), such as speakers , printers , etc . the computer 1102 may operate in a networked environment using logical connections through wired and / or wireless communications to one or more remote computers , such as a remote computer ( s ) 1148 . the remote computer ( s ) 1148 can be a workstation , a server computer , a router , a personal computer , portable computer , microprocessor - based entertainment appliance , a peer device or other common network node , and typically includes many or all of the elements described relative to the computer 1102 , although , for purposes of brevity , only a memory / storage device 1150 is illustrated . the logical connections depicted include wired / wireless connectivity to a local area network ( lan ) 1152 and / or larger networks , e . g ., a wide area network ( wan ) 1154 . such lan and wan networking environments are commonplace in offices and companies , and facilitate enterprise - wide computer networks , such as intranets , all of which may connect to a global communications network , e . g ., the internet . when used in a lan networking environment , the computer 1102 is connected to the local network 1152 through a wired and / or wireless communication network interface or adapter 1156 . the adaptor 1156 may facilitate wired or wireless communication to the lan 1152 , which may also include a wireless access point disposed thereon for communicating with the wireless adaptor 1156 . when used in a wan networking environment , the computer 1102 can include a modem 1158 , or is connected to a communications server on the wan 1154 , or has other means for establishing communications over the wan 1154 , such as by way of the internet . the modem 1158 , which can be internal or external and a wired or wireless device , is connected to the system bus 1108 through the serial port interface 1142 . in a networked environment , program modules depicted relative to the computer 1102 , or portions thereof , can be stored in the remote memory / storage device 1150 . it will be appreciated that the network connections shown are exemplary and other means of establishing a communications link between the computers can be used . the computer 1102 is operable to communicate with any wireless devices or entities operatively disposed in wireless communication , e . g ., a printer , scanner , desktop and / or portable computer , portable data assistant , communications satellite , any piece of equipment or location associated with a wirelessly detectable tag ( e . g ., a kiosk , news stand , restroom ), and telephone . this includes at least ieee - 802 . 11x ( wi - fi ), ieee - 802 . 15 ( bluetooth ™), and ieee - 802 . 16 ( wimax ) wireless technologies . thus , the communication can be a predefined structure as with a conventional network or simply an ad hoc communication between at least two devices . what has been described above includes examples of the various embodiments . it is , of course , not possible to describe every conceivable combination of components or methodologies for purposes of describing the various embodiments , but one of ordinary skill in the art may recognize that many further combinations and permutations are possible . accordingly , the subject specification intended to embrace all such alterations , modifications , and variations that fall within the spirit and scope of the appended claims . in particular and in regard to the various functions performed by the above described components , devices , circuits , systems and the like , the terms ( including a reference to a “ means ”) used to describe such components are intended to correspond , unless otherwise indicated , to any component which performs the specified function of the described component ( e . g ., a functional equivalent ), even though not structurally equivalent to the disclosed structure , which performs the function in the herein illustrated exemplary aspects . in this regard , it will also be recognized that the various aspects include a system as well as a computer - readable medium having computer - executable instructions for performing the acts and / or events of the various methods . in addition , while a particular feature may have been disclosed with respect to only one of several implementations , such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application . furthermore , to the extent that the terms “ includes ,” and “ including ” and variants thereof are used in either the detailed description or the claims , these terms are intended to be inclusive in a manner similar to the term “ comprising .” see , u . s . pat . no . 7 , 822 , 699 , expressly incorporated herein by reference . it should be understood that the various embodiments of the invention may be combined and subcombined in all possible consistent permutations . the scope of the invention is limited only by the claims , and no disclosed or preferred embodiment should be interpreted as limiting the generality of the invention . each of the below references is expressly incorporated herein by reference . j . khandurina , a . guttman , “ bioanalysis in microfluidic devices ”, j chromatography a ., vol . 943 , pp . 159 - 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