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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 4, Iss. 2 — Feb. 1, 2013
  • pp: 187–192
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Optical detection of nanoparticle-enhanced human papillomavirus genotyping microarrays

Xue Zhe Li, Sookyung Kim, Wonhyung Cho, and Seung-Yop Lee  »View Author Affiliations


Biomedical Optics Express, Vol. 4, Issue 2, pp. 187-192 (2013)
http://dx.doi.org/10.1364/BOE.4.000187


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Abstract

In this study, we propose a new detection method of nanoparticle-enhanced human papillomavirus genotyping microarrays using a DVD optical pick-up with a photodiode. The HPV genotyping DNA chip was labeled using Au/Ag core-shell nanoparticles, prepared on a treatment glass substrate. Then, the bio information of the HPV genotyping target DNA was detected by measuring the difference of the optical signals between the DNA spots and the background parts for cervical cancer diagnosis. Moreover the approximate linear relationship between the concentration of the HPV genotyping target DNA and the optical signal depending on the density of Au/Ag core-shell nanoparticles was obtained by performing a spot finding algorithm. It is shown that the nanoparticle-labeled HPV genotyping target DNA can be measured and quantified by collecting the low-cost photodiode signal on the treatment glass chip, replacing high-cost fluorescence microarray scanners using a photomultiplier tube.

© 2012 OSA

1. Introduction

Over the past decade, many advanced techniques have been developed to detect the bio information of various DNA, RNA and Protein chips. Typically biochips are prepared on a glass plate after they are labeled by fluorescence materials such as Cy3 or Cy5. Biochip microarrays have become extremely valuable tools in gene discovery, disease diagnosis, and drug discovery. After labeling, biochips are scanned using instruments specifically designed to detect fluorescent signals [1

1. M. J. Heller, “DNA microarray technology: devices, systems, and applications,” Annu. Rev. Biomed. Eng. 4(1), 129–153 (2002). [CrossRef] [PubMed]

,2

2. M. Schena, DNA Chip Analysis (Wiley, 2003).

]. There are two major technologies for fluorescence detection. One uses laser for excitation and photodetectors for detection, and the other uses a CCD or CMOS array for image collection [3

3. M. Schena, D. Shalon, R. W. Davis, and P. O. Brown, “Quantitative monitoring of gene expression patterns with a complementary DNA microarray,” Science 270(5235), 467–470 (1995). [CrossRef] [PubMed]

5

5. Y. Ardeshirpour, M. J. Deen, and S. Shirani, “Two-dimensional CMOS-based image sensor system for fluorescent detection,” Can. J. Electr. Comput. Eng. 29(4), 231–235 (2004). [CrossRef]

].

Conventional biochip detectors scan the fluorescent signals of spots with the different concentrations of dye, and then they analyze the fluorescence intensity to diagnose a disease. However this fluorescence-based detection technique suffers from weak emission signal, photochemical instability and environment-dependent quantum yield. Moreover the fluorescence-based biochip will not be able to keep a long time at room temperature due to degradation and instability [6

6. T. A. Taton, C. A. Mirkin, and R. L. Letsinger, “Scanometric DNA array detection with nanoparticle probes,” Science 289(5485), 1757–1760 (2000). [CrossRef] [PubMed]

]. Photodetectors for optical detection fundamentally operate on the transition of an electron from a lower energy state to a higher energy state as a result of the absorption of a photon.Most of commercial fluorescence scanners use a photomultiplier tube (PMT) for detection because it is the most sensitive photodetector in the visible light range [7

7. N. C. Tansil and Z. Gao, “Nanoparticles in biomolecular detection,” Nano Today 1(1), 28–37 (2006). [CrossRef]

]. However, a PMT is not the best choice for fluorescence detection because of its high operating voltages, fragility, size, and cost. An alternative approach employing wide field imaging such as CCD detectors has high quantum efficiency across the visible and near infrared and the ability to use broadband light sources.

In order to overcome the shortcomings of the fluorescence-based detection technique, many researcher adopt nanoparticles to label biomolecules instead of fluorescence dyes [6

6. T. A. Taton, C. A. Mirkin, and R. L. Letsinger, “Scanometric DNA array detection with nanoparticle probes,” Science 289(5485), 1757–1760 (2000). [CrossRef] [PubMed]

9

9. G. A. Blab, L. Cognet, S. Berciaud, I. Alexandre, D. Husar, J. Remacle, and B. Lounis, “Optical readout of gold nanoparticle-based DNA microarrays without silver enhancement,” Biophys. J. 90(1), L13–L15 (2006). [CrossRef] [PubMed]

]. The use of nanoparticles as labels in biomolecule detection in place of conventional molecular fluorophores has led to improvements in sensitivity, selectivity, and multiplexing capacity. For example, it is reported that DNA microarray detection with Ag-deposited Au labels using a typical fluorescent scanner has achieved a sensitivity that is 100 times greater than that of conventional fluorescence-based detection [6

6. T. A. Taton, C. A. Mirkin, and R. L. Letsinger, “Scanometric DNA array detection with nanoparticle probes,” Science 289(5485), 1757–1760 (2000). [CrossRef] [PubMed]

]. However, in nanoparticle-based optical detection, the nanoparticle layers and depositions lead to a complicated process for manufacturing the nanoparticle-based microarrays. Therefore, further simplification is needed to take these nanoparticle-based technologies from the laboratory to point of care [7

7. N. C. Tansil and Z. Gao, “Nanoparticles in biomolecular detection,” Nano Today 1(1), 28–37 (2006). [CrossRef]

]. Recently, the authors have developed a fluorescent detection scanner for DNA microarrays using a commercial DVD optical pick-up with low cost and small size [10

10. K.-H. Kim, S.-Y. Lee, S. Kim, and S. G. Jeong, “DNA microarray scanner with a DVD pick-up head,” Curr. Appl. Phys. 8(6), 687–691 (2008). [CrossRef]

,11

11. K.-H. Kim, S.-Y. Lee, S. Kim, S.-H. Lee, and S.-G. Jeong, “A new DNA chip detection mechanism using optical pick-up actuators,” Microsyst. Technol. 13(8-10), 1359–1369 (2007). [CrossRef]

]. However, even though the DVD pick-up module includes a photodiode, the fluorescence scanner uses a PMT rather than the built-in photodiode in order to amplify weak fluorescent signals. The optical pick-up module is also used to detect fluorescent profiles in microfluidic channels [12

12. L. I. Segerink, M. J. Koster, A. J. Sprenkels, and A. van den Berg, “A low-cost 2D fluorescence detection system for μm sized beads on-chip,” Lab Chip 12(10), 1780–1783 (2012). [CrossRef] [PubMed]

].

In this paper, we propose an optical detector of nanoparticle-based microarrays using a photodiode (PD) sensor rather than a PMT with high cost and large size. In order to verify the feasibility the photodiode based detector, we use Au/Ag core-shell nanoparticles to label human papillomavirus genotyping DNA. Then the bio information of the DNA molecules is quantified by comparing the photodiode signals measured at DNA spots and background regions on the HPV microarray.

2. System design

2.1. Design of optical detection system

Figure 1(a)
Fig. 1 (a) A schematic diagram of the DVD optical pick-up with photodiode sensor; (b) DVD pick-up used in experiments and its major components; (c) the experimental setup of photodiode based detection system
illustrates shows a schematic diagram of a DVD pick-up based detection system. The DVD optical pick-up is the commercial product (HPD-65) manufactured by Hitachi Electronics, as shown in Fig. 1(b). The optical detection system consists of a red laser diode, two photodiodes, a voice coil motor (VCM) and several optical parts including a collimating lens, a polarizing beam splitter and a dichroic filter. The numerical aperture of the focusing objective lens is 0.64 with a focal distance of 3.22 mm. The DVD optical pick-up has two laser diodes, one for reading compact discs (CD) and the other for reading denser DVD’s. The DVD laser diode generates red light with the wavelength of 650 nm.

There are two different optical paths with two PDs in the diagram. One is for auto-focusing by a four- quadrant PD and the other is for optical detection by an additional PD. Small part of the laser beam passing through the beam splitter is used to regulate the laser power by a front monitor sensor. Most of the laser beam is transmitted through the beam splitter, then reflected by the dichroic filter and focused onto the surface of the DNA microarray chip. A part of reflected beam from the chip surface passes through the dichroic filter and it is finally focused onto the four-quadrant PD. The focus error signal is calculated by the distribution of the beam spot across the four divided regions of the PD. The focus error signal is used to drive the VCM actuator in such a way that the objective lens is shifted to make light beam focused to the chip surface. The other part of laser beam reflected from the surface of the DNA chip is reflected from the surface of the dichroic filter. Then it is focused onto the additional PD sensor by an objective lens after the laser beam is reflected by the beam splitter. The analogue signal measured by the PD sensor is converted into the digital signal by an analogue digital converter. Finally, the digital signal is transmitted to a personal computer for analyzing the bio information of the DNA chip.

Figure 1(c) shows a photo of the experimental setup for the photodiode based microarray detection system. The rotary stage consists of a disk platform with slots loading DNA chips and a disk rotation motor. A maximum of eight slide glasses can be loaded on the disk platform, simultaneously. The rotation speed of the rotary stage is controlled by the disk rotation motor from 1 rpm to 600 rpm. A linear motor is used to move the optical pick-up detection module in the horizontal plane. The pick-up module moves along the radial direction of the rotary stage with the resolution on 2 ~10 μm as the stage rotates.

2.2. HPV genotyping microarray

The HPV genotyping DNA chip, labeled by Au/Ag core-shell nanoparticles for cervical cancer diagnosis, was prepared by the following process [17

17. D.-K. Lim, I.-J. Kim, and J.-M. Nam, “DNA-embedded Au/Ag core-shell nanoparticles,” Chem. Commun. (Camb.) 42(42), 5312–5314 (2008). [CrossRef] [PubMed]

]. First, the HPV genotyping immobilized probe DNA was spotted on a treatment glass chip by using the pin array method. Then, the HPV genotyping target DNA for the low risk 6, 11, 16 and 18 types was labeled by gold nanoparticles with a diameter of 13 nm and the concentration of the target DNA was changed from 0.05 pmol/μl to 0.5 pmol/μl. In order to improve the sensitivity of optical detection, the gold nanoparticles, which label the HPV genotyping target DNA, were enhanced to 15 nm in diameter by conjugating with silver. Finally, the target DNA was hybridized with the HPV genotyping immobilized probe DNA in one well on the treatment glass chip, as shown in Fig. 2(a)
Fig. 2 (a) A process diagram of the HPV genotyping microarray preparation; (b) the analysis software to load the scanned image and calculate the signal to background ratio of the HPV genotyping DNA with the low risk 6, 11, 16 and 18 types.
. The bio information of the HPV genotyping microarray can be quantified through collecting the optical signals with the wavelength of 650 nm which is reflected from the chip surface. The density of the labeled nanoparticles is proportional to the concentration of the target DNA. Therefore the photodiode signal depending on the labeled nanoparticles increases with the concentration of the target DNA.

3. Experiments and results

In order to analyze the optical signal measured by the PD based detection system, we define the signal and background ratio (SBR) as the ratio of the mean value of digital signal for spot to the mean value of digital signal for background:

SBR=Rs/Rb,
(1)

Here we simply select the low risk 11 type to appraise the detector system. The feasibility of the detector system is demonstrated by measuring the hybridized information of the HPV genotyping microarray for the low risk 11 type. In each DNA microarray, the region for the target DNA of 11 type consists of six spots which are marked with a blue circle in Fig. 2(b). Four spots are for the housekeeping beta-globin (HBB) DNA and two spots are for the target DNA of the low risk 11 type. The size of each spot is 200 μm. All the samples of the HPV genotyping microarrays with different concentrations of the target DNA of 0.05, 0.1, 0.25 and 0.5 pmol/μl are scanned by the photodiode based detection system. We execute the analysis software for each scanned image to calculate the SBR and the diagnostic result as shown in Fig. 3(b). The relationship between the concentration of the target DNA and the SBR are plotted with square markers in Fig. 3(c). The curve-fitting graph shows a good linear relationship between the concentration of the target DNA and the SBR.

However, the photodiode-measured optical signal is actually laser beam reflected from the nanoparticle-enhanced spot of the target DNA of the low risk 11. Since the HPV genotyping target DNA is labeled by Au/Ag core-shell nanoparticles, it is necessary to investigate the relationship between the concentration of the target DNA and the density of the labeled nanoparticles. The density of Au/Ag core-shell nanoparticles was measured by a scanning electron microscope (SEM). The SEM images for different concentrations of the target DNA (0.05, 0.1, 0.25 and 0.5 pmol/μl) are shown in Fig. 4
Fig. 4 SEM images of the Au/Ag core-shell nanoparticles for the HPV genotyping microarray with different concentrations of the target DNA (0.05, 0.1, 0.25 and 0.5 pmol/μl)
. From each SEM image, the number of the labeled nanoparticles was counted. The density of the labeled nanoparticles on the glass substrate were increased from 230 ea/μm2 to 1050 ea/μm2 as the concentration of the target DNA of the low risk 11 type was changed from 0.05 pmol/μl to 0.5 pmol/μl. It is considered that the target DNA labeled nanoparticles was well hybridized with the HPV genotyping immobilized probe DNA on the glass substrate. The relationship between the concentration of the target DNA and the density of the labeled nanoparticles is plotted in Fig. 3(c), together with the SBR. It is shown that the concentration of the target DNA is in good agreement with the density of the labeled nanoparticles.

4. Conclusion

In this study, we propose a photodiode-based detection method of nanoparticle-enhanced human papillomavirus genotyping microarrays for cervical cancer diagnosis. In order to overcome the signal degradation and instability of the fluorescence based detection techniques, we adopt the Au/Ag core-shell nanoparticles to label the HPV genotyping target DNA molecules. The nanoparticle-enhanced HPV microarrays with different concentrations of the target DNA are analyzed by measuring the optical signal reflected from the spot with the DNA information and the background part. Experimental results show that the concentration of the target DNA has linear relationships with both the signal-to-background ratio and the density of Au/Ag core-shell nanoparticles. The nanoparticle-labeled HPV genotyping target DNA can be simply quantified by collecting the low-cost photodiode signal reflected from the chip surface, replacing high-cost fluorescence microarray scanners using a photomultiplier tube.

Acknowledgments

This work is supported by the Ministry of Knowledge Economy (10035559) and the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (Grant No. 2010-0014728)

References and links

1.

M. J. Heller, “DNA microarray technology: devices, systems, and applications,” Annu. Rev. Biomed. Eng. 4(1), 129–153 (2002). [CrossRef] [PubMed]

2.

M. Schena, DNA Chip Analysis (Wiley, 2003).

3.

M. Schena, D. Shalon, R. W. Davis, and P. O. Brown, “Quantitative monitoring of gene expression patterns with a complementary DNA microarray,” Science 270(5235), 467–470 (1995). [CrossRef] [PubMed]

4.

G. Hamilton, N. Brown, V. Oseroff, B. Huey, R. Segraves, D. Sudar, J. Kumler, D. Albertson, and D. Pinkel, “A large field CCD system for quantitative imaging of microarrays,” Nucleic Acids Res. 34(8), e58–e72 (2006). [CrossRef] [PubMed]

5.

Y. Ardeshirpour, M. J. Deen, and S. Shirani, “Two-dimensional CMOS-based image sensor system for fluorescent detection,” Can. J. Electr. Comput. Eng. 29(4), 231–235 (2004). [CrossRef]

6.

T. A. Taton, C. A. Mirkin, and R. L. Letsinger, “Scanometric DNA array detection with nanoparticle probes,” Science 289(5485), 1757–1760 (2000). [CrossRef] [PubMed]

7.

N. C. Tansil and Z. Gao, “Nanoparticles in biomolecular detection,” Nano Today 1(1), 28–37 (2006). [CrossRef]

8.

A. J. Haes and R. P. Van Duyne, “A nanoscale optical biosensor: sensitivity and selectivity of an approach based on the localized surface plasmon resonance spectroscopy of triangular silver nanoparticles,” J. Am. Chem. Soc. 124(35), 10596–10604 (2002). [CrossRef] [PubMed]

9.

G. A. Blab, L. Cognet, S. Berciaud, I. Alexandre, D. Husar, J. Remacle, and B. Lounis, “Optical readout of gold nanoparticle-based DNA microarrays without silver enhancement,” Biophys. J. 90(1), L13–L15 (2006). [CrossRef] [PubMed]

10.

K.-H. Kim, S.-Y. Lee, S. Kim, and S. G. Jeong, “DNA microarray scanner with a DVD pick-up head,” Curr. Appl. Phys. 8(6), 687–691 (2008). [CrossRef]

11.

K.-H. Kim, S.-Y. Lee, S. Kim, S.-H. Lee, and S.-G. Jeong, “A new DNA chip detection mechanism using optical pick-up actuators,” Microsyst. Technol. 13(8-10), 1359–1369 (2007). [CrossRef]

12.

L. I. Segerink, M. J. Koster, A. J. Sprenkels, and A. van den Berg, “A low-cost 2D fluorescence detection system for μm sized beads on-chip,” Lab Chip 12(10), 1780–1783 (2012). [CrossRef] [PubMed]

13.

S. E. McFadden and L. Schumann, “The role of human papillomavirus in screening for cervical cancer,” J. Am. Acad. Nurse Pract. 13(3), 116–126, quiz 127–128 (2001). [CrossRef] [PubMed]

14.

F. X. Bosch and S. de Sanjosé, “Human papillomavirus in cervical cancer,” Curr. Oncol. Rep. 4(2), 175–184 (2002). [CrossRef] [PubMed]

15.

M. B. Miller and Y. W. Tang, “Basic concepts of microarrays and potential applications in clinical microbiology,” Clin. Microbiol. Rev. 22(4), 611–633 (2009). [CrossRef] [PubMed]

16.

H. J. An, N. H. Cho, S. Y. Lee, I. H. Kim, C. Lee, S. J. Kim, M. S. Mun, S. H. Kim, and J. K. Jeong, “Correlation of cervical carcinoma and precancerous lesions with human papillomavirus (HPV) genotypes detected with the HPV DNA chip microarray method,” Cancer 97(7), 1672–1680 (2003). [CrossRef] [PubMed]

17.

D.-K. Lim, I.-J. Kim, and J.-M. Nam, “DNA-embedded Au/Ag core-shell nanoparticles,” Chem. Commun. (Camb.) 42(42), 5312–5314 (2008). [CrossRef] [PubMed]

OCIS Codes
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(170.3890) Medical optics and biotechnology : Medical optics instrumentation
(170.4580) Medical optics and biotechnology : Optical diagnostics for medicine

ToC Category:
Biosensors and Molecular Diagnostics

History
Original Manuscript: September 10, 2012
Revised Manuscript: December 3, 2012
Manuscript Accepted: December 19, 2012
Published: December 20, 2012

Citation
Xue Zhe Li, Sookyung Kim, Wonhyung Cho, and Seung-Yop Lee, "Optical detection of nanoparticle-enhanced human papillomavirus genotyping microarrays," Biomed. Opt. Express 4, 187-192 (2013)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-4-2-187


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References

  1. M. J. Heller, “DNA microarray technology: devices, systems, and applications,” Annu. Rev. Biomed. Eng.4(1), 129–153 (2002). [CrossRef] [PubMed]
  2. M. Schena, DNA Chip Analysis (Wiley, 2003).
  3. M. Schena, D. Shalon, R. W. Davis, and P. O. Brown, “Quantitative monitoring of gene expression patterns with a complementary DNA microarray,” Science270(5235), 467–470 (1995). [CrossRef] [PubMed]
  4. G. Hamilton, N. Brown, V. Oseroff, B. Huey, R. Segraves, D. Sudar, J. Kumler, D. Albertson, and D. Pinkel, “A large field CCD system for quantitative imaging of microarrays,” Nucleic Acids Res.34(8), e58–e72 (2006). [CrossRef] [PubMed]
  5. Y. Ardeshirpour, M. J. Deen, and S. Shirani, “Two-dimensional CMOS-based image sensor system for fluorescent detection,” Can. J. Electr. Comput. Eng.29(4), 231–235 (2004). [CrossRef]
  6. T. A. Taton, C. A. Mirkin, and R. L. Letsinger, “Scanometric DNA array detection with nanoparticle probes,” Science289(5485), 1757–1760 (2000). [CrossRef] [PubMed]
  7. N. C. Tansil and Z. Gao, “Nanoparticles in biomolecular detection,” Nano Today1(1), 28–37 (2006). [CrossRef]
  8. A. J. Haes and R. P. Van Duyne, “A nanoscale optical biosensor: sensitivity and selectivity of an approach based on the localized surface plasmon resonance spectroscopy of triangular silver nanoparticles,” J. Am. Chem. Soc.124(35), 10596–10604 (2002). [CrossRef] [PubMed]
  9. G. A. Blab, L. Cognet, S. Berciaud, I. Alexandre, D. Husar, J. Remacle, and B. Lounis, “Optical readout of gold nanoparticle-based DNA microarrays without silver enhancement,” Biophys. J.90(1), L13–L15 (2006). [CrossRef] [PubMed]
  10. K.-H. Kim, S.-Y. Lee, S. Kim, and S. G. Jeong, “DNA microarray scanner with a DVD pick-up head,” Curr. Appl. Phys.8(6), 687–691 (2008). [CrossRef]
  11. K.-H. Kim, S.-Y. Lee, S. Kim, S.-H. Lee, and S.-G. Jeong, “A new DNA chip detection mechanism using optical pick-up actuators,” Microsyst. Technol.13(8-10), 1359–1369 (2007). [CrossRef]
  12. L. I. Segerink, M. J. Koster, A. J. Sprenkels, and A. van den Berg, “A low-cost 2D fluorescence detection system for μm sized beads on-chip,” Lab Chip12(10), 1780–1783 (2012). [CrossRef] [PubMed]
  13. S. E. McFadden and L. Schumann, “The role of human papillomavirus in screening for cervical cancer,” J. Am. Acad. Nurse Pract.13(3), 116–126, quiz 127–128 (2001). [CrossRef] [PubMed]
  14. F. X. Bosch and S. de Sanjosé, “Human papillomavirus in cervical cancer,” Curr. Oncol. Rep.4(2), 175–184 (2002). [CrossRef] [PubMed]
  15. M. B. Miller and Y. W. Tang, “Basic concepts of microarrays and potential applications in clinical microbiology,” Clin. Microbiol. Rev.22(4), 611–633 (2009). [CrossRef] [PubMed]
  16. H. J. An, N. H. Cho, S. Y. Lee, I. H. Kim, C. Lee, S. J. Kim, M. S. Mun, S. H. Kim, and J. K. Jeong, “Correlation of cervical carcinoma and precancerous lesions with human papillomavirus (HPV) genotypes detected with the HPV DNA chip microarray method,” Cancer97(7), 1672–1680 (2003). [CrossRef] [PubMed]
  17. D.-K. Lim, I.-J. Kim, and J.-M. Nam, “DNA-embedded Au/Ag core-shell nanoparticles,” Chem. Commun. (Camb.)42(42), 5312–5314 (2008). [CrossRef] [PubMed]

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