WO2010007350A1

WO2010007350A1 - Detections of microorganisms by dielectrophoresis.

Info

Publication number: WO2010007350A1
Application number: PCT/GB2009/001698
Authority: WO
Inventors: Peter Salmon; Robert Stewart Anthony
Original assignee: Blood Analysis Limited
Priority date: 2008-07-16
Filing date: 2009-07-09
Publication date: 2010-01-21
Also published as: EP2310133A1; GB0812999D0

Abstract

A method of collecting, detecting and enumerating microorganisms in a fluid comprising subjecting a sample of the fluid to dielectrophoresis and collecting the microorganisms onto a microelectrode, scanning the microelectrode using a scanning laser and determining the number of microorganisms present on the microelectrode.

Description

DETECTION OF MICROORGANISMS BY DIELECTROPHORESIS

The present invention relates to a method and apparatus for collecting, detecting and enumerating microorganism in fluids. In particular, it relates to the rapid detection and enumeration of microorganisms in mammalian fluids such as blood or blood products.

A previously used method for detecting and enumerating microbial contamination of blood or blood products involved culruring a sample of blood or blood product. However, such a method was too slow, requiring several hours/days incubation. It was also too insensitive to be of any practical use. Elder, A F et al found that up to 50% of bacterially contaminated platelets may escape detection by culture at 24 hours (see Transfusion, (2007), 47, 1134).

A solid phase laser scanner has been used to enumerate bacteria in water. Broadway, S C et al in Appl Environ Microbial. (2003), 69(7), 4272-4273 described the rapid staining and enumeration of small numbers of bacteria in water using solid-phase laser cytometry. In order to determine the number of bacteria, a sample of water was filtered through a black polycarbonate membrane and then an overlay of SYBR Green I dye was applied to the filter. After incubation, and removal of the stain, the membrane was dried and chilled prior to laser scanning. This method suffers from the disadvantage that interaction of the dye with the membrane filter produces non-specific spots of stain. In addition, microscopic examination of the membrane is usually necessary to identify possible non-specific stains. Other particulates in the water may take up the dye and become trapped on the membrane resulting in false positive counts.

Dielectrophoresis (DEP), which is the motion of electrically neutral particles or cells in response to a non-uniform electric field and can occur equally well in both DC and AC electric fields, has been used to quantify the number of particles in a liquid sample. Allsopp, D W E et al in J Phvs D: App Phvs. (1999), 32, 1066-1074 described an impedance technique for measuring dielectrophoretic collection of microbiological particles. The authors showed that measurement of the impedance change resulting from the collection of microbiological particles at coplanar electrodes enabled them to quantify the concentration of particles collected under positive dielectrophoretic force. The disadvantages of this method are a) low sensitivity in that at least 10⁵ml bacteria are required to incur a measurable impedance change, b) inflexible sample conditions in that the bacteria must be suspended in a buffer with an extremely low conductivity. Furthermore the change in impedance does not correlate with an accurate bacterial count. The size and cell wall characteristics influence the magnitude of the impedance change.

In order to safe-guard the biological safety of blood or blood products a rapid screening technique is needed that will detect less than 1,000 bacteria per ml of sample, preferably less than 100 bacteria per ml. Unfortunately, measuring impedance change is not sensitive enough and cannot detect such low levels of contamination. ■ . -.

It has now been found that by using dielectrophoresis microorganisms can be attracted out of a suspending fluid into the focal plane of a scanning laser and photomultiplier tubes to rapidly detect and quantify microbial contamination of the fluid.

According to one aspect of the invention, there is provided a method of collecting and detecting microorganisms in a fluid comprising the steps of subjecting a sample of said fluid to dielectrophoresis and collecting the microorganisms onto a microelectrode, scanning the microelectrode using a scanning laser and determining the number of microorganisms present on the microelectrode.

According to a second aspect of the invention, there is provided the use of dielectrophoresis in combination with laser scanning cytometry to collect and detect microorganisms in a fluid.

The method may be used for detecting microorganisms such as bacteria, viruses, yeasts, algae, protozoa and fungi. .

The fluid may be any mammalian fluid such as urine or cerebrospinal fluid, however, the method is particularly useful for detecting microorganisms in blood or blood products, such as platelets.

In order to separate any contaminating microorganisms, prior to subjecting the sample of fluid to dielectrophoresis, a lysis solution is preferably added in order to lyse any mammalian cells present. The contaminating microorganism may then be separated using centrifugation, after which they are stained or labelled so that they will fluoresce when subjected to the scanning laser. Suitable stains or labels may include non-specific nuclear dyes such as SYBR Green I and acridine orange, metabolic substrates that become fluorescent through enzymatic activity, antibodies, including monoclonal antibodies, to microbial proteins, or molecular probes which can hybridise to microbial genetic material or a combination of these. \

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings, in which:

Figure 1 shows the concept of DEP; Figure 2 shows an electrode placed in a laser scanner; and

Figure 3 shows an electrode in a well.

The labelled contaminating microorganisms suspended in a fluid are then loaded onto a microelectrode. The microelectrode comprises at least one pair of adjacent co-planar electrodes of micron dimensions for electrode width or gap size, and is supported on a substrate. The microelectrodes can be manufactured in various metals, including gold and aluminium. The substrate material is transparent and preferably of low autofluorescence, for example glass or plastic. The microelectrode may be manufactured by micro- fabrication technology employing photolithography or by printing metal ink technology or by printing and electrode plating technology or by a combination of these methods. The microelectrode may be placed in the bottom of a well, which may form part of a 96-well plate. In the well, the fluid remains static during DEP collection of the microorganism. Alternatively, the microelectrode may be placed in a flow through chamber where the suspending fluid containing the microorganisms is passed over the microelectrode on one side of the chamber in order to facilitate DEP collection from a larger sample. The microelectrode structure in these examples would normally be of a co- planar type. Alternatively, for a flowing sample, the microelectrode may be of a grid construction where a series of insulated grids are aligned to allow the passage of fluid.

Dielectrophoretic forces are produced across the microelectrode by an alternating current of fixed amplitude and wavelength. This may vary depending upon the conductivity of the suspending medium and the type of microorganisms to be collected. The signal may be a sine wave or a square wave and may or may not have a direct current offset depending upon the conductivity of the sample and the type of microorganism to be collected. DEP collects and concentrates any microorganisms suspended in the fluid onto the edge of the microelectrode. . .

Once the microorganisms have become trapped on the microelectrode, the current is switched off and the microelectrode is placed in the laser scanner. Scanning lasers and photomultiplier tube (PMT) detectors scan the area surrounding the microelectrode to excite the fluorescently labelled microorganisms and detect the emitted light. The wavelength used for laser scanning may vary depending on the fluorochrome used in the marker dye. In systems where two or more marker dyes of different excitation wavelengths are used, more than one source of laser light will be used.

The fluorescence intensity is collected at regular intervals by the PMTs and thresholding algorithms identify all the fluorescence intensities above background levels. Object intensity profiles enable the calculation of a range of morphological and fluorescent parameters to identify microorganisms collected onto the electrodes from the fluid sample. The advantages of using ^electrophoresis and scanning laser cytometry to detect microbial contamination of fluids, such as blood or blood products, are that the preparation technique is simple, several samples can be analysed simultaneously and a true real-time analysis of contamination can be obtained rapidly. Furthermore, no complex reagents are required and minimal waste is produced by virtue of using microelectrodes. Thus, a sensitive, high through-put real-time point of issue test for contamination is produced.

The present invention will be further described by way of reference to the following example. .

Example

Lysis solution (0.2 ml of 5% Triton, phosphate buffered saline (PBS) containing 10⁹ polyethylene imide (PEI) coated paramagnetic beads (10mm diameter)) was added to ^■ human blood platelets (1 ml) in a microfuge tube (2 ml). The tube was centrifuged using Eppendorf centrifuge 5424 at 20,00 xg for 2 min and then placed in a magnetic particle separator (mps). The beads and contaminating bacteria were allowed to collect. on the wall of the tube and the lysate supernatant was poured off. The tube was removed from the mps and staining solution (50 μl of SYBR green 1: 1,000 in 2 micro Si quarter strength Ringers solution) was added. The tube was incubated at room temperature for 5 minutes in the dark. The stained sample was the pipetted into a well in a dielectrophoretic microelectrode chip and connected to an alternating current (AC) signal of 100 KHz, 10 V amplitude for 10 minutes: The AC signal source was disconnected, the microelectrode chip was inverted in the scanning holder and loaded into a Bac-Detect laser scanner (Bac-Detect is available from Blood Analysis Ltd, PO BOX 71, Slough SL2 3SE). Laser scanning of the DEP collected bacterial was initiated. The results were displayed by the software as pass or fail depending on the level of bacterial contamination detected in the platelet sample. Alternatively they can be displayed as an exact bacterial count and the bacteria visualised by an image of then scanning surface.

Figure 1 shows DEP schematically. A sample A is passed across a substrate 1 having printed upon it, or otherwise formed upon it, a microelectrode structure comprising interdigitated electrodes 2 and 3. AC current 4 is generated and applied to the electrode via connections 5 and 6 to the respective electrodes 2 and 3. The electrodes are of micron dimensions and are energised with the voltage of a predetermined frequency using AC generator 4. The relevant particles (such as bacteria, biological cells and so on) collect on the electrode array and then, after the deposition process, the substrate can be analysed by visual inspection using microscopes or otherwise to count the number of particles and therefore information about the type and/or concentration of particles can be determined.

Figure 2 shows, again very schematically, a scanning laser 6 and focusing optics 7 focussing a laser beam 8 onto a microelectrode 9. The optics may be an integral part of the laser, or separate. Scanning means, for causing the beam to scan relative to the sample may be included.

DEP is used to collect microorganisms onto the edge of the electrode as discussed and the laser scans to detect these.

Figure 3 shows a microelectrode 9a within a well 10.

Claims

1. A method of collecting and detecting microorganisms in a fluid comprising the following steps: a) subjecting a sample of said fluid to dielectrophoresis and collecting the microorganisms onto a microelectrode; and b) scanning the microelectrode using a scanning laser and determining the number of microorganisms present on the microelectrode.

2. The method according to claim 1 which is used for detecting microorganisms such as bacteria, viruses, yeasts, algae, protozoa and fungi.

3. The method according to claim 1 or claim 2 wherein the sample of fluid is a . mammalian fluid selected from urine, cerebrospinal fluid, blood and blood products such as platelets. . . ^•

4. The method according to any one of claims 1 to 3 wherein, prior to subjecting the fluid sample to dielectrophoresis, a lysis solution is optionally added to the sample of fluid and any microorganisms present are separated by centrifugation.

5. The method according to any one of the preceding claims wherein, prior to subjecting the fluid sample to dielectrophoresis, the microorganisms present in the sample are stained or labelled.

6. The method according to claim 5 wherein the stain or label is selected from nonspecific nuclear dyes, metabolic substrates that become fluorescent through enzymatic activity, antibodies to microbial proteins and molecular probes that hybridise to microbial genetic material.

7. The method according to claim 1 wherein in step b) the microelectrode and area surrounding the microelectrode are scanned using the scanning laser connected to a photomultiplier tube detector to excite fluorescently labelled microorganisms and detect emitted light.

8. The method according to claim 7 wherein means are provided for enumerating and/or identifying microorganisms collected onto the microelectrode from the fluid sample.

9. Use of dielectrophoresis in combination with laser scanning cytometry to collect, detect and optionally enumerate microorganisms in a fluid.

10. Use according to claim 9 for detecting microorganisms such as bacteria, viruses, yeasts, algae, protozoa and fungi.

11. Use according to claim 9 or claim 10 wherein the fluid is a mammalian fluid selected from urine, cerebrospinal fluid, blood and blood products such as platelets.