Keywords: Drug Discovery; Biosensing; Crohn’s Disease; Hyperchromacity; Squeezed Light; Plasmonics
Abbreviations: FRET: Förster Resonance Energy Transfer; PBFRET: Photobleaching Resonance Energy Transfer; LSC: Laser Scanning Cytometry
Introduction
Those who ever used a stream-in-air flow cytometer and when
running their most interesting samples they just got an air bubble
trapped in the nozzle never dreamed of a time when this awful
situation just turned back showing its good face in helping to find
the best drug for curing a disease! The following story is somewhat
reminiscent of that of photobleaching, which was originally has
been discovered as a pure nuisence reducing visibility of fluorescent
samples, but with time it started to be used as a „kinetic ruler” for
probing phenomena mediating relaxation of the excited states
of dyes, leading to elaboration of methods like „photobleaching
fluorescence resonance energy transfer” or „pbFRET” [1]. The
central task of simultaneously probing thousands-millions of
protein-protein, protein-DNA interactions, posed by a demand for
solving biological complexity and problems of drug discovery has
been tackled by introducing a highly versatile family of bisosensing
technologies of outstandingly high speed, sensitivity and degree
of multiplexation, collectively termed as „high throughput-high
content (or capacity) screening assays, HT-HCS-A” [2]. Driven
largely by the development of microscopy and imaging techniques
(mainly confocal microscopy) different imaging solutions have been
introduced such as the different plate-readers in biochemistry.
In the field of cell biology, the analogue candidates are the
different types of cytometers (microscopes with moving objects),
basically slide-based or flow-based platforms, which inherently
have the demanded high-troughput-high capacity attributes,
originating from the large number of different (mainly optical)
signal channels (up to ~17 different colour fluorescence and light
scattering signals) and the high speed of cell detection (~103-104/
sec). While slide based cytometry (laser scanning cytometry, LSC)
is an excellent tool for monitoring adherent cells in their close tonative
environments, for non-adherent cells in suspension, flow
cytometry is the method of choice. Now, the problem at hand is
how to convert a flow cytometer traditionally used in a „single
sample-single run mode” into a device with wich different samples
can be measured at a 40-100 samples/minute rate, i.e. to find
the appropriate interface between an array of a huge number of
different cell samples and the flow cytometer! The answer to this
problem is given by the group of L. A. Sklar in New Mexico, in their
works aiming at converting flow cytometry into a potent tool for
drug discovery during the past decade. Experiencing initially
with and 8-pole flow switch - „plug cytometry” [3] -, their work
culminated in inventing a very ingenious sample „aspirating probe
and transfer” arrangement called HyperCyt platform [4-6].
The soal of the arrangement is a sliding probe capable of high speed motion in the x and y directions above a sample holder plate of wells, and alternatively sucking sample from the well or air between two subsequent wells, in succession, which are further transported towards the flow cytometer by a peristaltic pump. The end result is a train of samples from the respective wells with air bubbles separating them, each having relative lengths determined by the time periods spent by the probe in the well and in the air. Although this idea at first looks rather simple, its practical application demanded a lot of optimisation procedures regarding fitting of tubings of proper material and inner-diameter, peristaltic pump frequency (sample-air bubble frequency) to minimize particle carry-over between successive samples (if the tubings are not washed in between) and operating pressure of flow cytometers to avoid skewness of histograms belonging to the different samples (wells) occuring at such high sample speeds (~2 l/s), proper shaping of wells, and sample mixing by rotation to ensure sample homogenity [6,7].
In addition to that it can be conveniently fit to any flow cytometer, another remarkable feature of the HyperCyt platform is that it can be parallelized. In their work, B.S. Edwards, et al. [8] demonstrate the usage of HyperCyt when 4 probes connected to their respective cytometers are operated in tandem, allowing simultanous sampling from four parralel 384-well sections of a 1536-well plate taking only 11 minutes! By applying multiplexed bead and cell based assays, the authors demonstrate that also at these high sampling rates good quality data - as assessed by the „Z’ scores” significantly larger than 0.5 - can be obtained. After setting the stage for the realization of drug screening experiments in flow conditions, for what kind of assays can the new approch be used? Practically any cell constituent of physiological parameter - even dynamical parameters such as rotational motion (via fluorescence anisotropy) and proximity (via Förster transfer), i.e. molecular conformation – can be assayed depending on the availability of appropriate labels. Moreover signal-modulators (agonists and antagonists) can be screened in competition type assays like the nice illustrations of the authors: a G protein-coupled receptorrelated, Formylpeptide Receptor (FPR) ligand binding inhibition assay, a protease inhibition assay by which anthrax lethal factor can also be detected amongst others, and a G protein-coupled receptorkinase 2 (GRK2) enzyme inhibition assay. It should be noted here that, besides the high statistical power, another strength of flow cytometry is its high sensitivity, originating from its low background signal detection.
A promising approach to drug screening is when the physical screening process is carried out on a preselected collection of drugs and receptors structurally fitting into a classification scheme constructed in-silico based on previous experience on the behaviour (whether agonist or antagonist) and chemical structure of drugs and receptors („virtual screening”). After prefiltering by a „virtual screening” scheme, the „hit-rate” of finding drugs of the appropriate properties can be substantially increased, by >10-fold, as reported by the authors [9]. As opposed to measurement of ligand binding in equilibrium („primary” or „endpoint” assays), time-kinetical recordings of ligand binding („secondary” or „timed” assays) are also made possible by the HyperCyt system, during which cells are continually mixed („incubated”) with preformed coctail of reagents during the aspirating probe-cytometer sample delevery time. More detailed binding characteristics of drugs and their inhibitors, even the time course of the elicited cell-signaling events - e.g. dose response curves by realising preformed concentration gradients in the wells of the plate [5,7] - can be recorded this way with a time resolution determined by sample delivery and working frequency of the aspirating probe.
A proposed future application of this method would be e.g. when pair-wise proximity mapping of a set of receptors is made by measuring Förster Type of Resonance Energy Transfer (FRET) on preselected cell populations (different phenotypes, e.g. CD4+ or CD8+ T-cells, or CD4+CD25+ double-positive helper T, or Treg cells) of peripheral blood or surgical samples of persons suffering in different illnesses (e.g. Crohn’s disease, colorectal cancer). In this measuring scheme at least 3-4 different colours are needed: two colours for FRET donor and acceptor, and another one or two colours for gating out the necessary cell population(s) [10]. In this case the number of samples is „exponentially growing” with the number of receptors involved in the proximity mapping, due to the need for the donor-only and acceptor-only samples in addition to the double-labeled ones, a typical system amenable for multiplexing. This sampling technology is also amenable for a larger degree of multiplexing by applying new types of light emitters with sharper emission spectra - the „hyperchromacity principle” [11]. A future marriage with the emerging nanocrystal, nanolaser, and plasmonics technologies seem to be promising in this respect [12]. For slide and imaging based screening platforms the optical resolution is also decisive. New types of light sources - e.g. time- ordered squeezed light from a nonlinear crystal, with a reduced Poisson-noise [13] - offering larger spatial resolution are good candidates for developing more sensitive biosensors.
Acknowledgement
Financial support to L.B. for this work was provided by TÁMOP-4.2.2.A-11/1/KONV-2012-0045 project co-financed by the European Union and the European Social Fund, OTKA Bridging Fund support OSTRAT/810/213, and science financing support 1G3DBLR0TUDF-247 by the University of Debrecen.
References
- Bacsó Z, Bene L, Bodnár A, Matkó J, Damjanovich S (1996) A photobleaching energy transfer analysis of CD8/MHC-I and LFA-1/ICAM-1 interactions in CTL-target cell conjugates. Immunol Lett 54: 151-156.
- Nolan JP, Yang L (2007) The flow of cytometry into systems biology. Briefings in functional genomics and proteomics 6(2): 81-90.
- Edwards BS, Kuckuck FW, Sklar LA (1999) Plug flow cytometry: an automated coupling device for rapid sequential flow cytometric sample analysis. Cytometry 37: 156-159.
- Kuckuck FW, Edwards BS, Sklar LA (2001) High throughput flow cytometry. Cytometry 44: 83-90.
- Edwards BS, Kuckuck FW, Prossnitz ER, Ransom JT, Sklar LA (2001) HTPS flow cytometry: a novel platform for automated high throughput drug discovery and characterization. J Biomol. Screen 6: 83-90.
- Ramirez S, Aiken CT, Andrzejewski B, Sklar LA, Edwards BS (2003) High-throughput flow cytometry: validation in microvolume bioassays. Cytometry 53A: 55-65.
- Jackson WC, Kuckuck FW, Edwards BS, Mammoli A, Gallegos CM, et al. (2002) Mixing small volumes for continuous high-throughput flow cytometry: performance of a mixing Y and peristaltic sample delivery. Cytometry 47: 183-191.
- Edwards BS, Zhu JS, Chen J, Carter MB, Thal DM, et al. (2012) Cluster cytometry for high capacity bioanalysis. Cytometry 81A: 419-429.
- Edwards BS, Bologa Cristian, Young SM, Balakin KV, Prossnitz ER, et al. (2005) Integration of virtual screening with high-throughput flow cytometry to identify novel small molecule formylpeptide receptor antagonists. Mol Pharmacol 68: 1301-1310.
- Damjanovich L, Volkó J, Forgács A, Hohenberger W, Bene L (2012) Crohn's disease alters MHC-rafts in CD4+ T-cells. Cytometry 81A: 149-164.
- Mittag A, Lenz D, Gerstner AOH, Tárnok A (2006) Hyperchromatic cytometry principles for cytomics using slide-based cytometry. Cytometry 69: 691-703.
- Noginov MA, Zhu G, Belgrave AM, Bakker R, Shalaev VM, et al. (2009) Demonstration of a spaser-based nanolaser. Nat. Lett 460: 1110-1113.
- Bachor H-A, Ralph TC (2004) Applications of squeezed light. In: A guide to experiments in quantum optics. (2nd ,). Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA pp. 310-338.