We have developed a range of optical biochip devices for conducting live and fixed cell-based assays. The devices
encompass the ability to process an entire assay including fluorescently labelling cells, a microfluidic system to transport
and maintain cells to deliver them to an optical area of the device for measurement, with the possibility of a
incorporating a sorting step in between. On-chip excitation provided by red emitting LED and lasers define the excitation
wavelength of the fluorophore to be incorporated into the assay readout. The challenge for such an integrated
microfluidic optical biochip has been to identify and characterise a longterm fluorescent label suitable for tracking cell
proliferation status in living cells.
Traditional organic fluorophores have inherent disadvantages when considering their use for an on-chip device requiring
longterm cellular tracking. This has led us to utilise inorganic quantum dots (QDots) as fluorophores for on- chip assays.
QDs have unique properties such as photostability, broad absorption and narrow emission spectra and are available in a
range of emission wavelengths including far red. They also have much higher quantum efficiencies than traditional
organic fluorophores thus increasing the possible dynamic range for on-chip detection. Some of the QDots used have the
added advantage of labelling intact cells and being retained and distributed among daughter cells at division, allowing
their detection for up to 6 generations. The use of these QDs off-chip has suggested that they are ideal for live cell, nonperturbing
labelling of division events, whereby over time the QD signal becomes diluted with each generation.
Here we describe the use of quantum dots as live cell tracers for proliferating populations and the potential applications
in drug screening and optical biochip environments.
We demonstrate complete integration of a fluorescence-based assay in that the analyte well is also an optical emitter.
Laser machining is used to create 'active micro-wells' within semiconductor light emitting diode and laser structures.
These are then used to optically excite fluorescently-labelled beads in solution within the well. The results show
efficient illumination on a par with traditional lamp-based excitation. This technology therefore provides active microwell
plates with completely localized excitation, confined to the analysis well, that can be engineered via the micro-well
geometry. The micro-wells have also been machined within the cavity of lasing semiconductor structures and coherent
emission maintained. Thus lasing multi-well plates are also realizable.
An optical biochip is being developed for monitoring the sensitivity of biological cells to a range of environmental
changes. Such changes may include external factors such as temperature but can include changes within the suspending
media of the cell. The ability to measure such sensitivity has a broad application base including environmental
monitoring, toxicity evaluation and drug discovery. The device under development, capable of operating with both
suspension and adherent cell populations, employs electrokinetic processes to monitor subtle changes in the physicochemical
properties of cells as environmental parameters are varied. As such, the device is required to maintain cells in
a viable condition for extended periods of time.
The final device will employ integrated optical illumination of cells using red emitting LED or laser devices with light
delivery to measurement regions achieved using integrated micro-optical components. Measurements of electrokinetic
phenomena such as dielectrophoresis and electrorotation will be achieved through integrated optical detectors.
Environmental parameters can be varied while cells are actively retained within a measurement structure. This enables
the properties and sensitivity of a cell population to be temporally tracked.
The optical biochip described here uses a combination of microfabrication techniques including photolithographic and
laser micromachining processes. Here we describe the design and manufacturing processes to create the components of
the environmental monitoring strutures of the optical biochip.
We present details of the development of a optical biochip, with integrated on-chip laser excitation, for fluorescence
intensity cell based assays. The biochip incorporates an "active surface" for the control and manipulation of fluorescent
species placed directly on the device. The active elements of the biochip are one-dimensional periodic sub-wavelength
corrugations fabricated on a thin gold film. We have made fluorescence intensity measurements of both an organic dye
(Cy5), and immobilized and fluorescently labeled (with 705 nm emitting quantum dots), mammalian tumor cells in
contact with the active surface. Here we show that the presence of the periodic grating can be used to control both the
excitation and fluorescence generation process itself. We demonstrate that the gratings convert evanescent surface optical
modes into well-defined beams of radiation in the far-field and at the surface of the device this produces highly
contrasting regions of fluorescence excitation providing regions of high spatial selectivity.
Colloidal quantum dots (QDs) are now commercially available in a bio-functionalized form and Förster resonance
energy transfer (FRET) between bioconjugated dots and fluorophores within the visible range has been observed by
several groups of researchers. We are particularly interested in the far-red region, as from a biological perspective, there
are benefits in pushing to ~700 nm to minimize optical absorption (ABS) within tissue and avoiding cell
autofluorescence. We report on FRET between streptavidin (STV) conjugated CdTe quantum dots, Qdot705-STV, with
biotinylated Dy731-Bio fluorescent molecules in a donor-acceptor assay. We also highlight an unusual change in
Dy731-Bio absorptivity during the streptavidin-biotin binding process that can be attributed to the structural
reorientation. In moving to wavelengths beyond 700 nm, different alloy compositions are required for the quantum dot
core and these introduce associated changes in the physical shape. These changes directly affect the fluorescence decay
dynamics producing a marked biexponential decay with an extremely long lifetime component, a lifetime in excess of
100 ns. We compare and contrast the influence of the two QD relaxation processes upon the FRET dynamics in the
presence of Dy731-Bio.
In this paper we report on the development of an optical biochip to control both the excitation and resultant fluorescence
using grating coupled surface plasmons. Electron beam lithography is used to fabricate line gratings in thin layers of gold
on the surface of 150μm thick coverslips. Laser diodes operating at 630nm are close coupled to the coverslip resulting in
the excitation of surface plasmons. In the region of the grating light can radiate into the far-field, and both the angle of
emission and beam divergence can be controlled by the grating pitch and the number of lines included in the pattern. A
model is presented which treats the grating as an optical antenna array which shows how these characteristics can be
explained in terms of the wave vector matching between the surface plasmons and the grating. Fluorescence has also
been excited in standard organic dyes on-chip. When placed in close proximity to the surface of the sample strong
quenching of the fluorescence is seen in the region of the grating. In contrast an enhancement of the signal is seen when
the fluorophores are placed on a 200nm thick spacer layer.
In situ spectral analysis can be used to understand the targeting and interaction of agents in cellular compartments. A range of novel red excitable fluorescent probes, related to the anthraquinone family of anti-cancer agents, were designed for their DNA affinic properties and their ability to enter and penetrate living cells. We report on the spectral features of these probes, both in solution and bound within intact cells, to identify unique fluorescent signatures that exploit their use in bioassays on optical biochip devices.
The probes demonstrated red shifted emission spectra and increased 2 photon lifetime, with minimal fluorescent enhancement, upon binding to DNA. Spectral confocal laser scanning microscopy revealed complex emission profiles representing the bound (nuclear) and unbound (cytoplasmic) fractions of the DNA probes within live interphase, mitotic and apoptotic cells.
Analysis of the emission peaks encoded the spectra to provide cell compartment recognition and profiles for cells in different cell states. Sampling the entire emission spectra of these probes for cell locating, even in the presence of unbound molecules, provides good signal-to-noise in biochip devices. Furthermore, by sampling the fluorescence output at specific spectral windows we can obtain high spatial information without imaging.
The technological challenge is to integrate these fluorophores and appropriate detection capacity onto an optical biochip platform with microfluidic systems for cell handling.
We report on the development of a simple technique for obtaining time-domain information using dc detection of fluorescence. We show that this is feasible for assays where a change in lifetime of an indicator occurs in reaction to an analyte, in fluorescence resonance energy transfer for example, and could be particularly useful for assays performed in the scaled-down environment of a "lab-on-a-chip". A rate equation model is presented which allows an objective analysis of the relative importance of the key measurement parameters: optical saturation of the fluorophore and excitation pulse characteristics. We present a comparison of the model with a cuvette based analysis of a carbocyanine dye where the excitation source is a 650 nm wavelength, self-pulsing AlGaInP laser diode.
We report on the development of a stroboscopic excitation technique using a self-pulsing laser, and show that it is a feasible method for obtaining fluorescence lifetime information from a biochip format. The self-pulsing lasers described here are versatile devices which have been used for one photon excitation measurements to determine the lifetime of cyanine 5 in water and ethanol. The same devices have been used to develop a technique whereby the emphasis for time-resolution of a lifetime measurement can be transferred to the excitation source from the detector and processing electronics by virtue of the multiple-pulse, variable frequency nature of the laser output.