35
2.3.6.1 Fiber Optic DNA Biosensors
A general array protocol entails immobilizing a probe sequence (primer) that can
hybridize to its fluorescently-labeled complementary target. The fluorescent tag is
commonly incorporated into the target molecules via polymerase chain reaction
(PCR) [73]. This primer labeling method is convenient when amplification is needed
for detection, or when it is used to construct a cDNA library from a genomic RNA
pool via reverse transcription. In addition, the derivatized fiber core experiments can
detect unlabeled (non-fluorescent) target solutions [66]. This is achieved by
competitive hybridization with fluorescent target samples. In this method, the
fluorescent synthetic target complements are synthesized and initially hybridized to
the array to saturate the array probe elements [66]. The unlabeled target solution is
then hybridized to the same array, competing with the prehybridized synthetic
targets. The presence of the unlabeled target is determined by a fluorescence
decrease caused by the displacement of the fluorescent synthetic target by the
unlabeled species. This procedure eliminates the need to incorporate fluorescence
into the target and allows quantitative measurements to be performed. These fiber
optic platforms are the basis for microsphere array designs that improved array
fabrication and allowed extremely high-density sensor placement [66].
2.3.6.2 Analysis Setup and Protocol
The imaging system consists of a light source, an inverted microscope, and a modified Olympus epifluorescence microscope/charge-coupled device camera
(Photometrics PXL). A fiber chuck carried the imaging fiber in a fixed position
while electronically controlled filter wheels switched between the analytical
wavelength and the encoding wavelengths, enabling complete analysis and
identification of the microspheres within minutes. Excitation light is sent into the
proximal tip of the imaging fiber, and emission from the fluorescing molecules captured and directed onto the CCD camera detector (Fig. 2.18) [72A].
The multiplex analysis images were acquired for 1 and 0.5 s at wavelengths specific to each encoding dye. A 365-nm excitation filter and a 600-nm long-pass emission filter were used for the Eu-dye. A 620-nm excitation filter and a 670-nm
emission filter were used for the Cy5 dye. A 530-nm excitation filter and a 580-nm
emission filter were used for TAMRA. That is the detection of amplified DNA
fragments that were incubated with a fluorescein-labeled sequencing primer in the
existence of the two allelic ROX-ddNTP or TAMRA-ddNTP terminators. All targets
were labeled with fluorescein. It was shown that the fluorescence intensity was
proportional to the extent of hybridization at each probe position [72A]. In addition,
the camera was equipped with an internal chip that provides megapixel resolution
(1280/1024). This megapixel chip was able to resolve the arrays miniatured feature
sizes (3 mm) and provided multiple pixels for each optical channel in the fiber
bundle (Fig. 2.19).
2 Detection of Biological Warfare Agents Using Biosensors
2.3.6.1 Fiber Optic DNA Biosensors
A general array protocol entails immobilizing a probe sequence (primer) that can
hybridize to its fluorescently-labeled complementary target. The fluorescent tag is
commonly incorporated into the target molecules via polymerase chain reaction
(PCR) [73]. This primer labeling method is convenient when amplification is needed
for detection, or when it is used to construct a cDNA library from a genomic RNA
pool via reverse transcription. In addition, the derivatized fiber core experiments can
detect unlabeled (non-fluorescent) target solutions [66]. This is achieved by
competitive hybridization with fluorescent target samples. In this method, the
fluorescent synthetic target complements are synthesized and initially hybridized to
the array to saturate the array probe elements [66]. The unlabeled target solution is
then hybridized to the same array, competing with the prehybridized synthetic
targets. The presence of the unlabeled target is determined by a fluorescence
decrease caused by the displacement of the fluorescent synthetic target by the
unlabeled species. This procedure eliminates the need to incorporate fluorescence
into the target and allows quantitative measurements to be performed. These fiber
optic platforms are the basis for microsphere array designs that improved array
fabrication and allowed extremely high-density sensor placement [66].
2.3.6.2 Analysis Setup and Protocol
The imaging system consists of a light source, an inverted microscope, and a modified Olympus epifluorescence microscope/charge-coupled device camera
(Photometrics PXL). A fiber chuck carried the imaging fiber in a fixed position
while electronically controlled filter wheels switched between the analytical
wavelength and the encoding wavelengths, enabling complete analysis and
identification of the microspheres within minutes. Excitation light is sent into the
proximal tip of the imaging fiber, and emission from the fluorescing molecules captured and directed onto the CCD camera detector (Fig. 2.18) [72A].
The multiplex analysis images were acquired for 1 and 0.5 s at wavelengths specific to each encoding dye. A 365-nm excitation filter and a 600-nm long-pass emission filter were used for the Eu-dye. A 620-nm excitation filter and a 670-nm
emission filter were used for the Cy5 dye. A 530-nm excitation filter and a 580-nm
emission filter were used for TAMRA. That is the detection of amplified DNA
fragments that were incubated with a fluorescein-labeled sequencing primer in the
existence of the two allelic ROX-ddNTP or TAMRA-ddNTP terminators. All targets
were labeled with fluorescein. It was shown that the fluorescence intensity was
proportional to the extent of hybridization at each probe position [72A]. In addition,
the camera was equipped with an internal chip that provides megapixel resolution
(1280/1024). This megapixel chip was able to resolve the arrays miniatured feature
sizes (3 mm) and provided multiple pixels for each optical channel in the fiber
bundle (Fig. 2.19).
2 Detection of Biological Warfare Agents Using Biosensors