- Email: [email protected]
Software-based quantitation of bioassays on CD
Sensors and Actuators B 148 (2010) 620–623
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Short communication
Software-based quantitation of bioassays on CD Manu Pallapa a,b , Lily M.L. Ou a , M. (Ash) Parameswaran b,∗ , Hua-Zhong Yu a,∗∗ a b
Department of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada School of Engineering Science, Simon Fraser University, Burnaby, BC V5A 1S6, Canada
a r t i c l e
i n f o
Article history: Received 6 January 2010 Received in revised form 4 May 2010 Accepted 19 May 2010 Available online 27 May 2010 Keywords: Compact disc Biomarker detection Error detection IsoBuster
a b s t r a c t Compact discs (CDs) can be used as rapid, low-cost, high-capacity screening platforms for running bioassays with no modification of the detection hardware (conventional standard optical drive). We describe herein a new protocol to read and quantitate biotin–streptavidin binding assays with a standard optical drive by using a CD-data analysis software (IsoBuster), which identifies erroneous sectors by locating the exact error position bit-by-bit and allows various data formats to be used. The numbers of erroneous sectors increase as a function of the streptavidin concentration in the tested solutions. High spatial accuracy and detection sensitivity (0.3 g/mL) were achieved. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The compact disc (CD) has been explored as an assaying platform for extremely low-volume biological and chemical analyses; an optical drive can be adapted to serve as a signal readout device for biomarker detection. In 2000, Kido et al. reported the preparation of disc-based high-density immunoassay microarrays via piezoelectric inkjet printing, for which the signal readout relied on a conventional fluorescence scanner [1]. They also proposed the use of an unmodified optical drive to examine reflective immunobeadbased sandwich assays on CD [1]. Alexandre et al. [2] developed a colorimetric method for the detection of multiparametric DNA on a specially prepared “bioCD” (“storing” DNA hybridization assays on the external and numeric information on the inner portion of the same disc). The signals were detected with a custom-made doublesided CD reader for simultaneous analysis of both genomic and numerical data [2]: the spatial resolution was 300 m. Barathur et al. from Burstein Technologies Inc. (BTI) developed their version of “BioCompact Disc (BCD)”, an enclosed disc format read by a modified CD drive [3]. The key feature of their system is the sample analysis in the liquid phase within an enclosed chamber on the disc, which is performed concurrently with optical tracking of information features on the disc.
∗ Corresponding author. Tel.: +1 778 782 4971; fax: +1 778 782 4951. ∗∗ Corresponding author. Tel.: +1 778 782 8062; fax: +1 778 782 3765. E-mail addresses: [email protected] (M. Parameswaran), [email protected] (H.-Z. Yu). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.05.045
Recently, Lange et al. introduced a silver staining method (with antibody-labelled gold nanoparticles promoting the precipitation of silver particles) to increase the reflectivity of C-reactive protein (CRP) immunoassays on disc, which can be imaged with a CD reading (pickup) head mounted on an optical microscope stage [4]. With an analog signal acquisition approach, i.e., making electrical connections to obtain a differential signal from the optical photodiode component, Potyrailo et al. have shown the application of conventional CD/DVD drives for quantitative chemical sensing of metal cations [5]. Other researchers have explored software-based approaches to analyze the digital signals obtained from a CD. La Clair et al. reported an error determination routine for screening ligand-protein interactions on CDs with a specially designed software; they created unique data structures to detect reading errors by comparing the original with the retrieved data byte by byte [6]. Jones employed CD drives as photonic signal processing devices (optical microscopes) to image stained bacterial cells physically adsorbed on disc [7]. We have also developed a method for reading error numbers per frame of pre-recorded audio files on a CD-R [8]. Herein we examine the feasibility of using a commercial CD-data analysis software (e.g., IsoBuster) to read and quantitate biochemical binding assays prepared on a disc. The versatility of IsoBuster provides the advantage of using any data format/media. IsoBuster also allows the detection to be more specific; it identifies the erroneous bits rather than the total error numbers per frame. This provides the most “direct” approach to use consumer electronic products for biomedical diagnostics.
M. Pallapa et al. / Sensors and Actuators B 148 (2010) 620–623
621
Fig. 1. Surface patterning and assay preparation using PDMS microfluidic channel plates. The figure is not to scale.
2. Experimental 2.1. Reagents 1-Ethyl-3-(3 -dimethylaminopropyl)carbodiimide (EDC), Nhydroxysuccinimide (NHS), 2-(N-morpholino)ethanesulfonic acid (MES), bovine serum albumin (BSA), Tween 20 and gelatine were purchased from Sigma–Aldrich. Sodium chloride and phosphates were from Caledon Laboratories Ltd. Sodium azide was purchased from BioShop Canada Inc. EZ-link amine-PEG2-biotin was purchased from Thermo Scientific. The nanogold–streptavidin conjugate (1.4 nm in diameter) and LI silver enhancement kit were purchased from Nanoprobes. Deionized water (>18.3 M cm) from a Barnstead EasyPure UV/UF system (Dubuque, IA) was used to prepare the sample solutions. 2.2. Preparation of biotin–streptavidin binding assays on CD For the surface activation the polycarbonate side of the written CD was irradiated with UV light in the presence of ozone for 20 min. The UV/ozone cleaner (Model PSD-UV) was from Novascan Technologies, Inc. This UV/ozone treatment generates a high density of carboxylic acid groups on the CD surface which facilitates the attachment of amino-modified probe molecules such as aminated biotin to the CD surface via amide coupling [9]. Due to the mildness of the immobilization, the CD can still be read by a conventional optical drive. Before the immobilization of biotin molecules, the CD was treated with EDC (100 mM) and NHS (25 mM) prepared in a MES solution (0.1 M, pH 5.8) for 4 h. Surface patterning and bioassay preparation were carried out by microfluidic delivery of the amino-terminated biotin molecules and the target streptavidin samples (Fig. 1) via two polydimethylsiloxane (PDMS) channel plates (prepared using the Sylgard 184 Silicone Elastomer Kit). The elastomer base and curing agent were mixed in a 6:1 ratio by weight and the mixture was then poured into a silicon moulding master. After introducing 60 L of the MES solution containing biotin molecules (4 mM) into the opening (24 mm × 8 mm), the CD was incubated at room temperature overnight. Another PDMS stamp with microchannels (1 mm × 28 mm) was then used for the delivery of 10 L of the streptavidin–nanogold conjugate solution of various
concentrations in each channel. The testing solution was made in a phosphate buffer (20 mM) containing 0.8% BSA. After the binding reaction, the CD was incubated at room temperature for 1 h. The enhancement of the reading signal was accomplished by promoting the deposition of silver particles of up to a few hundred nm size [8]. 2.3. Software-based detection procedure Being stored in the form of pits and lands in a CD, digital information is read spirally from the centre outward by a laser in an optical drive. These pits and lands form the basis of binary codes referred to as bits. Eight bits constitute a byte, which is another commonly used unit for digital information. A frame, the basic unit of information, is comprised of 24 bytes of audio data, 8 bytes of parity bits, 3 bytes of sync data, and 1 byte of sub-code bits in an audio CD. A sector is a group of 98 frames [8]. Audio data was recorded on RiDATA Silver-Silver 700 MB CD-Rs with Nero 7 (Ultra Edition). A single audio file of 5 MB size repeated 60 times was written on each of two CDs for a set of experiments, one for the detection and the other as a reference to compare the hexadecimal data of the erroneous sectors with clean sectors. In each case, at least five different concentrations of streptavidin–gold nanoconjugate were tested. IsoBuster was used as the software to detect and quantitate the bioassays on CDs. This is a data recovery tool compatible with multi-file systems, multi-hard media (CD/DVD/BD/HD DVD), and multi-soft media (various CD and DVD data formats), which provides the freedom to use any data format/media to conduct biomolecule detection [10]. Though other CD scanning softwares have the ability to acquire the block error rate from media, they only count error frames, but not to the byte-by-byte details (as can be done with the single sector extraction utility of IsoBuster). It performs surface scans on hard media to check for physical reading errors which are identified and saved as a list of erroneous files. This utility is further exploited by the single sector extraction function which enables viewing of the exact sector and its erroneous data. The sector viewer gives the comprehensive logical block address to pinpoint the erroneous sector to the resolution of individual bits. This feature displays the hexadecimal data of a particular
622
M. Pallapa et al. / Sensors and Actuators B 148 (2010) 620–623
sector, allowing a comparison of the erroneous with reference data. 3. Results and discussion Biotin is a vitamin with strong affinity for streptavidin, a protein found in Streptomyces avidinii bacteria. As a tetramer, streptavidin forms a -barrel having four biotin binding sites in the interior of the barrel. The association between biotin and streptavidin is one of the strongest non-covalent interactions, which has a formation constant (Ka ) of approximately 1015 M−1 [6]. The feature of biotin–streptavidin association and affinity is often exploited as a diagnostic tool (model system) in biochemical assays. As mentioned in the experimental section, the biotin–streptavidin binding assays on CDs were prepared via a microfluidic approach; for the purpose of proof-of-concept, we have tested five different concentrations (from 0.32 to 1.60 g/mL) on each disc together with a negative control in which no biotin was deposited (illustrated in Fig. 2). Thus a six-line binding assay was generated for the software-based reading, i.e., six binding “strips” aligned radially outward with ascending concentrations. As shown in Fig. 2, visual shading is evident upon silver staining [8]. IsoBuster includes an option to create a list of all erroneous sectors of a file, track session or the entire medium, displaying only those that were truly read. The comprehensive procedure that we
Fig. 2. Optical photo showing the biotin–streptavidin binding “strips” prepared on a CD; the concentrations are given in g/mL.
employed included a full surface scan followed by extraction of the raw data from an erroneous file. This flags the affected tracks as erroneous (Fig. 3a). The figure shows five columns indicating the name of the track, logical block address (LBA), size of the track in
Fig. 3. Digital reading results of the bioassays on CD with concentration range of (0.32–1.60) g/mL (shown in Fig. 2). (a) Grouped errors detected by IsoBuster on three binding “strips”. (b) Error report showing the total number of errors caused by all binding “strips”.
M. Pallapa et al. / Sensors and Actuators B 148 (2010) 620–623
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4. Conclusion Utilizing the IsoBuster software and an unmodified optical drive, we have been able to quantitate the classical bioassays prepared on standard CD-Rs. A clear dependence of reading errors (number of erroneous sectors) on concentration was observed, which shows the quantitative capability of this software-based protocol. The detection is based on a comparison of the raw hexadecimal data of the erroneous sectors with the clean sectors of the same logical block address. This technique exhibits the ability to detect the exact locations of the binding assays on the disc and to correlate the errors with sample concentrations. Particularly IsoBuster provides the advantage of acquiring the positional and numeric quantization of the errors resulting from the binding “strips”, and the freedom to use data formats other than audio data. Further experiments are in progress to set up a high-density microarray for biomolecular or pathogen detection, which may eventually lead to a low-cost, rapid screening technology. Acknowledgement Fig. 4. Number of error sectors versus concentration of streptavidin–gold nanoparticle conjugates repeated over the course of three independent CD’s. The concentration range is 0.32–1.6 g/mL.
localized bytes, size of the track in bytes and the date when the original track was written. The tracks affected by errors are marked with a red “X” symbol. Each group of X’s corresponds to an individual strip; due to the width of each strip, several tracks were affected when a binding strip was formed on CD. The software also lists the total number of errors occurred. For the disc tested with a target concentration range of 0.32–1.6 g/mL (as shown Fig. 2), a total of 1508 erroneous sectors were detected (Fig. 3b). To ascertain the exact number of sectors affected and their locations, the written tracks were subjected to an error extraction function. This function scans through the affected track and creates a list of erroneous sectors, and gives options of extracting the sectors with or without raw hexadecimal data. Particularly when the function encounters an erroneous sector, it allows four possible actions: (1) omit any substitution data of affected sector; (2) replace the entire sector with zeros while keeping the sector size intact. With this option the sector containing the original data affected with errors will be changed to zeros; (3) replace only user data in the sector affected with errors with zeroes, implying that the raw data (sync bytes, headers, subheaders, etc.) are intact and the user data contain all zeroes; (4) replace the entire sector data including both the RAW and user data with hexadecimal error/sense codes, which inform the host (computer and/or application) of an error situation. In our experiments, the option (4) was selected. As a verification step, the Sector View (showing hexadecimal data) of the erroneous sector and the sector with the same logical block address on the reference CD was compared and showed data change. Comparison of unaffected sectors on the CDs with biotin–streptavidin binding assays and the reference CD, having the same logical block address (LBA) showed no change in hexadecimal data. The number of error sectors affected in a group (corresponding to a specific binding “strip”) and the concentration of streptavidin were plotted as shown in Fig. 4. The graph shows a clear monotonic increase as the concentration of target streptavidin increases. This result was reproducible on independently prepared CDs with this trial binding assay. Most importantly, the reading can be done with a conventional optical drive without any modification to either the hardware or the software driver, thus augmenting the potential applications in running bioassays with consumer electronic devices.
We wish to thank Dr. Eberhard Kiehlmann for reviewing the manuscript and to express our appreciation to Drs. Yunchao Li and Honglun Wang for their help in some of the experiments. This research was supported by funds from Natural Science and Engineering Research Council of Canada. References [1] H. Kido, A. Maquieira, B.D. Hammock, Disc-based immunoassay microarrays, Anal. Chim. Acta 411 (2000) 1–11. [2] I. Alexandre, Y. Houbion, J. Collet, S. Hamels, J. Demarteau, J.-L. Gala, J. Remacle, Compact disc with both numeric and genomic information as DNA microarray platform, BioTechniques 33 (2002) 435–439. [3] R. Barathur, J. Bookout, S. Sreevatsan, J. Gordon, M. Werner, G. Thor, M. Worthington, New disc-based technologies for diagnostic and research applications, Psychiatr. Genet. 12 (2002) 193–206. [4] S.A. Lange, G. Roth, S. Wittemann, T. Lacoste, A. Vetter, J. Grassle, S. Kopta, M. Kolleck, B. Breitinger, M. Wick, J.K. Heinrich Horber, S. Dubel, A. Bernard, Measuring biomolecular binding events with a compact disc player device, Angew. Chem. Int. Ed. 45 (2006) 270–273. [5] R.A. Potyrailo, W.G. Morris, A.M. Leach, T.M. Sivavec, M.B. Wisnudel, S. Boyette, Analog signal acquisition from computer optical disk drives for quantitative chemical sensing, Anal. Chem. 78 (2006) 5893–5899. [6] J.J. La Clair, M.D. Burkart, Molecular screening on a compact disc, Org. Biomol. Chem. 1 (2003) 3244–3249. [7] C.L. Jones, Cryptographic hash functions and CD-based optical biosensors, Probl. Nonlinear Anal. Eng. Syst. 2 (2005) 17–36. [8] Y. Li, L.M.L. Ou, H.-Z. Yu, Digitized molecular diagnostics: reading disk-based bioassays with standard computer drives, Anal. Chem. 80 (2008) 8216–8223. [9] Y. Li, Z. Wang, L.M.L. Ou, H.-Z. Yu, DNA detection on plastic: surface activation protocol to convert polycarbonate substrates to biochip platforms, Anal. Chem. 79 (2007) 426–433. [10] IsoBuster version 2.5, Smart Projects, 2008 and accompanying documentation (http://www.isobuster.com).
Biographies Manu Pallapa received his Bachelor’s degree in electrical and electronics engineering from University Visvesvaraya College of Engineering, Bangalore, India in 2006. He is currently pursuing the M.A.Sc. degree in MEMS engineering at Simon Fraser University. Lily M.L. Ou received her Bachelor’s degrees at Simon Fraser University in 2007, double majoring in Chemistry and Molecular Biology and Biochemistry. She is now a M.Sc. candidate in Dr. Yu’s research laboratory. Dr. Ash Parameswaran completed his doctoral degree in microelectronics in 1990 at the University of Alberta (Canada). He is now a professor at Simon Fraser University and heads an applied research group in MEMS. Dr. Hua-Zhong (Hogan) Yu is a professor in the Department of Chemistry at Simon Fraser University (Canada). His current research is focused on the surface chemistry of biochips and on the development of novel electronic and diagnostic devices.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Short communication
Software-based quantitation of bioassays on CD Manu Pallapa a,b , Lily M.L. Ou a , M. (Ash) Parameswaran b,∗ , Hua-Zhong Yu a,∗∗ a b
Department of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada School of Engineering Science, Simon Fraser University, Burnaby, BC V5A 1S6, Canada
a r t i c l e
i n f o
Article history: Received 6 January 2010 Received in revised form 4 May 2010 Accepted 19 May 2010 Available online 27 May 2010 Keywords: Compact disc Biomarker detection Error detection IsoBuster
a b s t r a c t Compact discs (CDs) can be used as rapid, low-cost, high-capacity screening platforms for running bioassays with no modification of the detection hardware (conventional standard optical drive). We describe herein a new protocol to read and quantitate biotin–streptavidin binding assays with a standard optical drive by using a CD-data analysis software (IsoBuster), which identifies erroneous sectors by locating the exact error position bit-by-bit and allows various data formats to be used. The numbers of erroneous sectors increase as a function of the streptavidin concentration in the tested solutions. High spatial accuracy and detection sensitivity (0.3 g/mL) were achieved. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The compact disc (CD) has been explored as an assaying platform for extremely low-volume biological and chemical analyses; an optical drive can be adapted to serve as a signal readout device for biomarker detection. In 2000, Kido et al. reported the preparation of disc-based high-density immunoassay microarrays via piezoelectric inkjet printing, for which the signal readout relied on a conventional fluorescence scanner [1]. They also proposed the use of an unmodified optical drive to examine reflective immunobeadbased sandwich assays on CD [1]. Alexandre et al. [2] developed a colorimetric method for the detection of multiparametric DNA on a specially prepared “bioCD” (“storing” DNA hybridization assays on the external and numeric information on the inner portion of the same disc). The signals were detected with a custom-made doublesided CD reader for simultaneous analysis of both genomic and numerical data [2]: the spatial resolution was 300 m. Barathur et al. from Burstein Technologies Inc. (BTI) developed their version of “BioCompact Disc (BCD)”, an enclosed disc format read by a modified CD drive [3]. The key feature of their system is the sample analysis in the liquid phase within an enclosed chamber on the disc, which is performed concurrently with optical tracking of information features on the disc.
∗ Corresponding author. Tel.: +1 778 782 4971; fax: +1 778 782 4951. ∗∗ Corresponding author. Tel.: +1 778 782 8062; fax: +1 778 782 3765. E-mail addresses: [email protected] (M. Parameswaran), [email protected] (H.-Z. Yu). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.05.045
Recently, Lange et al. introduced a silver staining method (with antibody-labelled gold nanoparticles promoting the precipitation of silver particles) to increase the reflectivity of C-reactive protein (CRP) immunoassays on disc, which can be imaged with a CD reading (pickup) head mounted on an optical microscope stage [4]. With an analog signal acquisition approach, i.e., making electrical connections to obtain a differential signal from the optical photodiode component, Potyrailo et al. have shown the application of conventional CD/DVD drives for quantitative chemical sensing of metal cations [5]. Other researchers have explored software-based approaches to analyze the digital signals obtained from a CD. La Clair et al. reported an error determination routine for screening ligand-protein interactions on CDs with a specially designed software; they created unique data structures to detect reading errors by comparing the original with the retrieved data byte by byte [6]. Jones employed CD drives as photonic signal processing devices (optical microscopes) to image stained bacterial cells physically adsorbed on disc [7]. We have also developed a method for reading error numbers per frame of pre-recorded audio files on a CD-R [8]. Herein we examine the feasibility of using a commercial CD-data analysis software (e.g., IsoBuster) to read and quantitate biochemical binding assays prepared on a disc. The versatility of IsoBuster provides the advantage of using any data format/media. IsoBuster also allows the detection to be more specific; it identifies the erroneous bits rather than the total error numbers per frame. This provides the most “direct” approach to use consumer electronic products for biomedical diagnostics.
M. Pallapa et al. / Sensors and Actuators B 148 (2010) 620–623
621
Fig. 1. Surface patterning and assay preparation using PDMS microfluidic channel plates. The figure is not to scale.
2. Experimental 2.1. Reagents 1-Ethyl-3-(3 -dimethylaminopropyl)carbodiimide (EDC), Nhydroxysuccinimide (NHS), 2-(N-morpholino)ethanesulfonic acid (MES), bovine serum albumin (BSA), Tween 20 and gelatine were purchased from Sigma–Aldrich. Sodium chloride and phosphates were from Caledon Laboratories Ltd. Sodium azide was purchased from BioShop Canada Inc. EZ-link amine-PEG2-biotin was purchased from Thermo Scientific. The nanogold–streptavidin conjugate (1.4 nm in diameter) and LI silver enhancement kit were purchased from Nanoprobes. Deionized water (>18.3 M cm) from a Barnstead EasyPure UV/UF system (Dubuque, IA) was used to prepare the sample solutions. 2.2. Preparation of biotin–streptavidin binding assays on CD For the surface activation the polycarbonate side of the written CD was irradiated with UV light in the presence of ozone for 20 min. The UV/ozone cleaner (Model PSD-UV) was from Novascan Technologies, Inc. This UV/ozone treatment generates a high density of carboxylic acid groups on the CD surface which facilitates the attachment of amino-modified probe molecules such as aminated biotin to the CD surface via amide coupling [9]. Due to the mildness of the immobilization, the CD can still be read by a conventional optical drive. Before the immobilization of biotin molecules, the CD was treated with EDC (100 mM) and NHS (25 mM) prepared in a MES solution (0.1 M, pH 5.8) for 4 h. Surface patterning and bioassay preparation were carried out by microfluidic delivery of the amino-terminated biotin molecules and the target streptavidin samples (Fig. 1) via two polydimethylsiloxane (PDMS) channel plates (prepared using the Sylgard 184 Silicone Elastomer Kit). The elastomer base and curing agent were mixed in a 6:1 ratio by weight and the mixture was then poured into a silicon moulding master. After introducing 60 L of the MES solution containing biotin molecules (4 mM) into the opening (24 mm × 8 mm), the CD was incubated at room temperature overnight. Another PDMS stamp with microchannels (1 mm × 28 mm) was then used for the delivery of 10 L of the streptavidin–nanogold conjugate solution of various
concentrations in each channel. The testing solution was made in a phosphate buffer (20 mM) containing 0.8% BSA. After the binding reaction, the CD was incubated at room temperature for 1 h. The enhancement of the reading signal was accomplished by promoting the deposition of silver particles of up to a few hundred nm size [8]. 2.3. Software-based detection procedure Being stored in the form of pits and lands in a CD, digital information is read spirally from the centre outward by a laser in an optical drive. These pits and lands form the basis of binary codes referred to as bits. Eight bits constitute a byte, which is another commonly used unit for digital information. A frame, the basic unit of information, is comprised of 24 bytes of audio data, 8 bytes of parity bits, 3 bytes of sync data, and 1 byte of sub-code bits in an audio CD. A sector is a group of 98 frames [8]. Audio data was recorded on RiDATA Silver-Silver 700 MB CD-Rs with Nero 7 (Ultra Edition). A single audio file of 5 MB size repeated 60 times was written on each of two CDs for a set of experiments, one for the detection and the other as a reference to compare the hexadecimal data of the erroneous sectors with clean sectors. In each case, at least five different concentrations of streptavidin–gold nanoconjugate were tested. IsoBuster was used as the software to detect and quantitate the bioassays on CDs. This is a data recovery tool compatible with multi-file systems, multi-hard media (CD/DVD/BD/HD DVD), and multi-soft media (various CD and DVD data formats), which provides the freedom to use any data format/media to conduct biomolecule detection [10]. Though other CD scanning softwares have the ability to acquire the block error rate from media, they only count error frames, but not to the byte-by-byte details (as can be done with the single sector extraction utility of IsoBuster). It performs surface scans on hard media to check for physical reading errors which are identified and saved as a list of erroneous files. This utility is further exploited by the single sector extraction function which enables viewing of the exact sector and its erroneous data. The sector viewer gives the comprehensive logical block address to pinpoint the erroneous sector to the resolution of individual bits. This feature displays the hexadecimal data of a particular
622
M. Pallapa et al. / Sensors and Actuators B 148 (2010) 620–623
sector, allowing a comparison of the erroneous with reference data. 3. Results and discussion Biotin is a vitamin with strong affinity for streptavidin, a protein found in Streptomyces avidinii bacteria. As a tetramer, streptavidin forms a -barrel having four biotin binding sites in the interior of the barrel. The association between biotin and streptavidin is one of the strongest non-covalent interactions, which has a formation constant (Ka ) of approximately 1015 M−1 [6]. The feature of biotin–streptavidin association and affinity is often exploited as a diagnostic tool (model system) in biochemical assays. As mentioned in the experimental section, the biotin–streptavidin binding assays on CDs were prepared via a microfluidic approach; for the purpose of proof-of-concept, we have tested five different concentrations (from 0.32 to 1.60 g/mL) on each disc together with a negative control in which no biotin was deposited (illustrated in Fig. 2). Thus a six-line binding assay was generated for the software-based reading, i.e., six binding “strips” aligned radially outward with ascending concentrations. As shown in Fig. 2, visual shading is evident upon silver staining [8]. IsoBuster includes an option to create a list of all erroneous sectors of a file, track session or the entire medium, displaying only those that were truly read. The comprehensive procedure that we
Fig. 2. Optical photo showing the biotin–streptavidin binding “strips” prepared on a CD; the concentrations are given in g/mL.
employed included a full surface scan followed by extraction of the raw data from an erroneous file. This flags the affected tracks as erroneous (Fig. 3a). The figure shows five columns indicating the name of the track, logical block address (LBA), size of the track in
Fig. 3. Digital reading results of the bioassays on CD with concentration range of (0.32–1.60) g/mL (shown in Fig. 2). (a) Grouped errors detected by IsoBuster on three binding “strips”. (b) Error report showing the total number of errors caused by all binding “strips”.
M. Pallapa et al. / Sensors and Actuators B 148 (2010) 620–623
623
4. Conclusion Utilizing the IsoBuster software and an unmodified optical drive, we have been able to quantitate the classical bioassays prepared on standard CD-Rs. A clear dependence of reading errors (number of erroneous sectors) on concentration was observed, which shows the quantitative capability of this software-based protocol. The detection is based on a comparison of the raw hexadecimal data of the erroneous sectors with the clean sectors of the same logical block address. This technique exhibits the ability to detect the exact locations of the binding assays on the disc and to correlate the errors with sample concentrations. Particularly IsoBuster provides the advantage of acquiring the positional and numeric quantization of the errors resulting from the binding “strips”, and the freedom to use data formats other than audio data. Further experiments are in progress to set up a high-density microarray for biomolecular or pathogen detection, which may eventually lead to a low-cost, rapid screening technology. Acknowledgement Fig. 4. Number of error sectors versus concentration of streptavidin–gold nanoparticle conjugates repeated over the course of three independent CD’s. The concentration range is 0.32–1.6 g/mL.
localized bytes, size of the track in bytes and the date when the original track was written. The tracks affected by errors are marked with a red “X” symbol. Each group of X’s corresponds to an individual strip; due to the width of each strip, several tracks were affected when a binding strip was formed on CD. The software also lists the total number of errors occurred. For the disc tested with a target concentration range of 0.32–1.6 g/mL (as shown Fig. 2), a total of 1508 erroneous sectors were detected (Fig. 3b). To ascertain the exact number of sectors affected and their locations, the written tracks were subjected to an error extraction function. This function scans through the affected track and creates a list of erroneous sectors, and gives options of extracting the sectors with or without raw hexadecimal data. Particularly when the function encounters an erroneous sector, it allows four possible actions: (1) omit any substitution data of affected sector; (2) replace the entire sector with zeros while keeping the sector size intact. With this option the sector containing the original data affected with errors will be changed to zeros; (3) replace only user data in the sector affected with errors with zeroes, implying that the raw data (sync bytes, headers, subheaders, etc.) are intact and the user data contain all zeroes; (4) replace the entire sector data including both the RAW and user data with hexadecimal error/sense codes, which inform the host (computer and/or application) of an error situation. In our experiments, the option (4) was selected. As a verification step, the Sector View (showing hexadecimal data) of the erroneous sector and the sector with the same logical block address on the reference CD was compared and showed data change. Comparison of unaffected sectors on the CDs with biotin–streptavidin binding assays and the reference CD, having the same logical block address (LBA) showed no change in hexadecimal data. The number of error sectors affected in a group (corresponding to a specific binding “strip”) and the concentration of streptavidin were plotted as shown in Fig. 4. The graph shows a clear monotonic increase as the concentration of target streptavidin increases. This result was reproducible on independently prepared CDs with this trial binding assay. Most importantly, the reading can be done with a conventional optical drive without any modification to either the hardware or the software driver, thus augmenting the potential applications in running bioassays with consumer electronic devices.
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Biographies Manu Pallapa received his Bachelor’s degree in electrical and electronics engineering from University Visvesvaraya College of Engineering, Bangalore, India in 2006. He is currently pursuing the M.A.Sc. degree in MEMS engineering at Simon Fraser University. Lily M.L. Ou received her Bachelor’s degrees at Simon Fraser University in 2007, double majoring in Chemistry and Molecular Biology and Biochemistry. She is now a M.Sc. candidate in Dr. Yu’s research laboratory. Dr. Ash Parameswaran completed his doctoral degree in microelectronics in 1990 at the University of Alberta (Canada). He is now a professor at Simon Fraser University and heads an applied research group in MEMS. Dr. Hua-Zhong (Hogan) Yu is a professor in the Department of Chemistry at Simon Fraser University (Canada). His current research is focused on the surface chemistry of biochips and on the development of novel electronic and diagnostic devices.