Incorporation of barrier and filter layers
Since the a-Si:H layer has a significant photosensitivity in the UV, a UV filter consisting of an a-SiC:H (Eg = 2.1 eV) layer was deposited above the a-Si:H photoconductor to lower its absorption of light at the PyMPO excitation wavelength. This increased the sensitivity of the device by one order of magnitude (Fig. , circles). It is estimated that the PyMPO absorbs about 0.4% of the light at 400 nm. A thin SiNx layer was used to reduce the spill-over of photogenerated carriers in the a-SiC:H into the a-Si:H. By adding the SiNx and a-SiC:H layers on top of the initial a-Si:H photodetector, the ratio between the device response at 560 and 400 nm increased from 3, for the initial a-Si:H single layer device (Fig. , squares) to 700 (Fig. , triangles).
Figure 2 Improvement of the sensitivity of the photodetector at 400 nm (λexc of PyMPO) by successively adding an a-SiC:H UV light filter layer, a SiNx carrier barrier layer and an interference filter. The spectral response curves were normalized to the (more ...)
To further increase the sensitivity of the device at 400 nm, an interference filter composed of a high-refractive-index quarter wave layer, followed by a multilayer of 15 bilayers of (λ/4) of SiOx (n = 1.57; d = 637 Å)/SiNx (n = 1.89; d = 529 Å) was fabricated. This filter reflects 95% of the light at 400 nm. The use of this type of filter above the detector/barrier/a-SiC:H filter stack increases the detector sensitivity by three orders of magnitude (Fig. , crossed circles). It is estimated that the filter stack allows ~60% of the light emitted by the PyMPO at 560 nm to reach the a-Si:H photodetector.
Improvements in the sensitivity of the a-Si:H photodetector device will require the development of a more effective filter to the UV light. High quality, precision bandpass interference filters can allow a rejection by a factor of 103 to 104 relative to the peak transmittance. This would allow the signal of the present device in the blue region to be decreased further by 3–4 orders of magnitude and would allow detection levels comparable to the presently used fluorescence techniques (e.g. laser scanning, CCD camera).
DNA calibration curve
Measurement of the SR curve of the detector in the presence of PyMPO shows an increase in the detector signal in the 400–450 nm range. This occurs because PyMPO, when excited at this wavelength, emits light with a spectrum centered in the visible (λem = 565 nm), which is in turn absorbed by the a-Si:H photoconductor, resulting in a photocurrent. Because the detector has an integrated UV filter, the overall effect of the system in the presence of UV illumination is an apparent increase in the response at 400–450 nm when PyMPO, or DNA labeled with PyMPO, is present on the surface. A system of filters, integrated in the chip between the a-Si:H photodetector and the SiO2 layer upon which the DNA probes are immobilized, serves to block the UV/blue excitation light while allowing the emitted light at 565 nm to be transmitted to the a-Si:H layer, producing a photoresponse. Thus, tagging DNA molecules with the dye PyMPO allows the use of an a-Si:H photodetector to optoelectronically detect the presence of DNA and to quantify the amount of labeled DNA present. To improve the signal to noise ratio of the device, it is crucial that the photoresponse of the sensor be as low as possible in the 400–450 nm excitation range.
In order to construct the DNA calibration curve, different amounts of DNA probes and targets labeled with PyMPO were adsorbed onto the optimized photodetector and the corresponding responses at 400 nm were measured. After washing away the probes, the spectral response of the a-Si:H photodetector returns to the initial state (Fig. , inset). The normalized response of the device at 400 nm is plotted in Figure for different concentrations of adsorbed DNA probe and target. Figure shows that the detection limit of the present device is ~2 pmol/cm2.
Figure 3 Photodetector calibration. The correlation between the photodetector normalized response at 400 nm and the surface densities of the DNA probe and target is shown. Calibration data obtained independently by fluorescence microscopy is shown for comparison. (more ...)
Calibration curves, using fluorescence microscopy as the detection method, were independently obtained for both DNA probe and target single-strand sequences (as described above). Figure shows that both the optoelectronic and fluorescence calibration curves have the same linear trend for the range of DNA surface densities studied.
Immobilized DNA density
The photodetector was used to quantify the surface density of DNA probes covalently immobilized on the functionalized film deposited over the detector layers. Figure shows that when labeled DNA is immobilized on the silanized SiO2
surface, there is an increase in the device response around 400 nm. Combining this response with the optoelectronic calibration curve of the DNA probe (Fig. , open circles), the immobilized DNA density is estimated to be 33.5 ± 4.0 pmol/cm2
. A comparable value is obtained when the DNA density is determined by fluorescence microscopy (31.5 ± 12.1 pmol/cm2
). This surface density falls within the typical 1–200 pmol/cm2
range reported in the literature for DNA immobilized on functionalized glass and SiO2
Figure 4 Normalized spectral response of the photodetector in its initial state, after silanization and crosslinking, after DNA immobilization with DNA containing the specific -SH termination that allows immobilization (ss-DNA-SH), and after exposure to DNA with (more ...)
A linear, single stranded, DNA molecule orientated perpendicularly to a surface can be assumed to occupy a cylindrical region in space with a diameter close to the diameter of a B DNA double helix (≈20 Å). Thus, an estimation of the theoretical coverage of a closely packed full monolayer of probes yields 53 pmol/cm2
. The density of the immobilized 5′-thiolated DNA probes reported above is typical of a high probe density regime (10
). It corresponds to about 63% of this theoretical full DNA monolayer coverage, and to ~4% of the silanols believed to be present in a clean SiO2
The 5′-end specificity of the DNA probe upon immobilization to the surface was confirmed by repeating the immobilization experiment under the same experimental conditions, but using a DNA probe devoid of the thiol group at the 5′-end. Figure shows that a significant optoelectronic and fluorescence signal is detected only if probes are thiolated at the 5′-end. This result confirms that DNA probes are immobilized on the chip surface only through the 5′-end of the probe and discards any other immobilization mechanism such as adsorption as responsible for the increase in detector signal.
DNA hybridization density
The preparation of surfaces with bound DNA probes in microarrays should guarantee that (i) DNA targets could easily access the probes and (ii) that the hybridization event is selective enough to enable discrimination between fully complementary and non-complementary targets. These concerns were addressed in the current biochip structure by comparing the level of hybridization attained with complementary and non-complementary DNA targets. First, unlabeled DNA capture probes were immobilized on the surface above the photodetector/filter system. Subsequently, non-complementary (negative control) and complementary (positive control) PyMPO-labeled DNA targets were incubated at the surface as described above and the optoelectronic (at 400 nm) and fluorescence responses were recorded. The results are shown in Figure .
Figure 5 Normalized spectral response of the bio-detector in its initial state (with immobilized untagged ssDNA), after pre-hybridization with BSA, and after hybridization of complementary and non-complementary ssDNA targets. The inset shows the signals obtained (more ...)
Hybridization with a complementary DNA target resulted in an increase in the photodetector signal at 400 nm, while non-complementary DNA targets yielded a negligible increase in signal. The density of the hybridized DNA determined with the photodetector was 3.7 ± 1.5 pmol/cm2
and 0.20 ± 0.07 pmol/cm2
for the positive and negative controls, respectively. These densities are in agreement with the values obtained by fluorescence microscopy for hybridization with complementary (6.3 ± 1.5 pmol/cm2
) and non-complementary (0.50 ± 0.075 pmol/cm2
) DNA sequences. The values obtained with the positive control are also in good agreement with data reported in the literature for DNA hybridization on silanized surfaces (11
). A comparison of the surface densities determined for the immobilized and hybridized DNA show that ~11% of the covalently attached probes participate in the formation of a double helix with the targets (hybridization efficiency). This low hybridization efficiency is typical of high probe density regimes (11
A DNA chip structure that integrates a functionalized SiO2 thin-film flat surface with a thin-film, multi-layered, optoelectronic detector based on a-Si:H was developed for the immobilization and hybridization of DNA. The device, which relies on the labeling of DNA molecules with the fluorophore PyMPO, can detect surface concentrations down to 2 × 1012 molecules/cm2. A linear detection behavior in the range of 2.5–700 pmol DNA/cm2 was observed. DNA probes were covalently immobilized on the chip surface via a thiol group and a surface density of 33.5 ± 4.0 pmol/cm2 was measured with the integrated photodetector. Furthermore, the hybridization of complementary DNA targets with immobilized and unlabeled DNA probes was successfully photodetected at a surface density of 3.7 ± 1.5 pmol/cm2.
The integrated DNA chip described in this paper presents important advantages over other optical systems: (i) there is no need for expensive and complex equipment for data acquisition and analysis; (ii) on-chip electronic data acquisition can be easily implemented; and (iii) the speed and the reliability of DNA chip hybridization pattern analysis can be improved. This detection system could form the basis of an optoelectronic detector array for the rapid, reliable and inexpensive detection of nucleic acids in a wide variety of DNA microarray applications. It points toward faster, reliable and less expensive biological data acquisition and can allow the use of DNA chips for clinical point-of-care.