- Filling in liquid without precision requirements
With this method, droplets can be released at the entrance of the well and will fill into the wells by capillary forces alone if the well surface is hydrophilic. In the event that the well surface is hydrophobic, there is a maximum droplet volume to well diameter size condition that needs to be satisfied. Nevertheless, this is amenable to efforts to reduce sample/reagent volume in HTS. After filling, the liquid column typically remains stationary even if the bottom end of the well is open. A challenge in this new approach is to understand and improve the efficacy with which samples/reagents can fill the capillary wells. Apart from precision relaxation, this method is extremely amenable in the drive towards use of smaller volumes for testing and offers greater immunity from splashing contamination due to isolation features offered within the capillary.
T.W. Ng, Y. Yu, H.Y. Tan, A. Neild, Capillary wells microplate, Appl. Phys. Lett. 93, 174105 (2008).
- Harnessing the point spread function for better sensitivity
The capillary wells approach offers advantages over standard microplates when applied in the imaging mode. This is due to the point spread function effect in most imaging lenses being more highly resolved in the lateral as opposed to axial sense. The imaging mode offers advantages of low magnification for initial screening, which can be followed up by high magnification for specific inspection.
H.Y. Tan, T.W. Ng, A. Neild, O.W. Liew, Point spread function effect in image-based fluorescent microplate detection. Analytical Biochemistry. 397 (2009) 256-258.
- Getting a side view in imaging measurement
The presence of bubbles in liquid samples residing in microplate wells cause inaccuracies in fluorescence measurements. In addition, pipetting errors, if not adequately managed, can result in misleading data and wrong interpretations of assay results; particularly in the context of high throughput screening. In this work, we describe an adapted design to the capillary wells microplate approach that permits side viewing. We demonstrate that it detects bubbles and pipetting errors during actual assay runs to ensure accuracy in screening.
H.Y. Tan, T.W. Ng, A. Neild, O.W. Liew Capillary wells microplate with side optical access. Journal of Biomolecular Screening. Accepted 10 May 2010.
- Basis of non filter non-imaging fluorometric measurement
Measurements can also be accomplished using a non-imaging mode. We have developed a novel approach for fluorometry with light-emitting-diodes (LEDs) as excitation source that capitalizes on the principle of light shielding and total internal reflection to operate without optical filters. This scheme is practicable, demonstrated to significantly reduce the amount of excitation light overlapping the fluorescence signals at the photodetector, operated best with excitation light applied orthogonal to detection, and permitted higher measurement sensitivity of fluorophore emission.
H.Y. Tan, T.W. Ng, O.W. Liew, Filterless fluorometry with enhanced sensitivity, Journal of Fluorescence. 19 (2009) 375-379.
- Using the sides in non-imaging measurement
In some situations, the non-imaging mode offers better flexibilities in measurement. We have shown that with some minor modifications, the non-imaging mode sensing with the capillary wells is possible. This has the capability to achieve both fluorescence and absorbance measurements. In addition, the features of determining the presence of bubbles and pippeting error have also been retained.
H.Y. Tan, B.H.P. Cheong, A. Neild, O.W. Liew, T.W. Ng Absorbance and fluorometric sensing with capillary wells microplates Review of Scientific Instruments. 84 (2010) 124301.
- Mixing by pushing out into a droplet and redrawing
Sample/reagent mixing is important in microplate instrumentation and the approach of mixing prior to dispensation into the wells can be problematic when assays involve a mixture of different proportions of reagents. We demonstrated with capillaries, a simple and highly effective method that uses air actuation and liquid surface tension to create pendant drops in which the inherent inner circulatory flow within accomplishes mixing [3]. The approach portends the capability to be incorporated into microplates in order to provide a useful feature for assay investigations.
J.K.K. Lye, T.W. Ng, A. Neild, O.W. Liew A capacity for mixing in capillary wells for microplates. Analytical Biochemistry. 410 (2011) 152-154.
- Droplet exposure in air allows pre-concentraion by evaporation
The preconcentration of analytes is important in biochemical analysis as it offers the ability to detect for trace species, and increase signal-to-noise ratios when using optical sensing on fluorophores. A strong advantage of the evaporation technique lies in its ability to operate without the need of any energy source; albeit major challenges exist on how to increase the surface area exposure to air for heightened evaporation, ensure no further increases once specified analyte concentrations have been achieved, and not needing any intervening membranes. We demonstrate here that the droplet creation and retraction approach in capillary based microplates offers such abilities whilst at the same time facilitating mixing.
F. Shao, T.W.Ng, J.K.K. Lye, O.W. Liew Evaporative pre-concentration of fluorescence in capillary wells microplates Journal of Fluorescence. Accepted 01 May 2011.
- Overcoming the liquid filling problem with standard microplates
It is often assumed that droplets dispensed into standard microplate wells will automatically fill their bottom. We show by computational simulation and experimental verification that the ability to fill the well bottom is dependent on the surface wetting characteristics. The release of droplets at the center was also found to fill the well bottom better than droplet dispensation in contact with the well wall. Hydrophobic surfaces required higher liquid volumes to fill the well bottom; unlike the case with capillary wells microplates. This renders standard microplate wells less amenable for use in small volume liquid handling.
G. Lu, H.Y. Tan, A. Neild, O.W. Liew, Y. Yu, T.W. Ng, Liquid filling in standard circular well microplates. Journal of Applied Physics. 108 (2010) 124701.
- Measuring the wettability of reagents
In biochemical response studies, pharmaceutically acceptable co-solvents or surfactants are typically employed to increase solubility. The low solubility in aqueous media displayed by many drugs is a known limitation to oral, parenteral, and transdermal administration. Polymeric micelles made by surfactants have a whole set of unique characteristics, which make them very promising carriers for a wide range of drugs. In high throughput drug formulation screening, verification that the correct amount of surfactant(s) has been introduced is important during formulation preparation. Here, we propose that this can be achieved by interrogating the reagent’s wettability whilst the reagent is present in its holder. This is especially important as small reagent volumes are increasingly used. Such a mode ensures that a) less time is needed to transfer liquid for tests, and b) reagent loss is minimized. For the latter it is important to note that any transfer of liquid out for testing will result in inevitable losses, and that externally tested volumes are never returned to the source due to contamination concerns. We also uncovered that illumination delivered from the liquid end of the capillary lights up the interface, thereby facilitating the measurement.
B.H.-P. Cheong, T.W. Ng, Y. Yu, O.W. Liew, Using the meniscus in a capillary for small volume contact angle measurement in biochemical applications. Langmuir. Accepted 23/08/2011.
See Video - Model of capillary filling guides preconcentration (NEW)
An attractive advantage of the capillary well microplate approach is the ability to conduct evaporative analyte preconcentration. The use of hydrophobic materials for the wells which apart from reducing material loss through wetting also affords self entry into the well when the droplet size reduces below a critical value. Using Surface Evolver simulation without gravity, we find the critical diameters fit very well with theoretical results. When simulating the critical diameters with gravity included, the gravitational effect could only be ignored when the liquid volumes were small (difference of 5.7% with 5mL of liquid), but not when the liquid volumes were large (differences of more than 22% with 50mL of liquid). From this, we developed a modifying equation from a series of simulation results made to describe the gravitational effect. In simulating the condition of multiple wells underneath each droplet, we found that having more holes did not alter the critical diameters significantly. Consequently, the modifying relation should also generally express the critical diameter for multiple wells under a droplet.
Y. Yu, X. Wang, T.W. Ng, Modeling the liquid filling in capillary well microplates for analyte preconcentration. Journal of Colloid and Interface Science. Accepted 7/3/2012.
See Video
Major Research Thrusts
Biochemical Analysis TechniquesWe seek to develop better methods, assays, and procedures that enable analysis of substances found in living organisms and the chemical reactions underlying life processes.
Particle/Cell ManipulationOur quest to find novel methods to move particles and cells around to accomplish important functions.
Instrumentation DevelopmentThe natural outcome of experimentation where we produce more advanced sensors, actuators, processors, and software.