WO2007146677A2

WO2007146677A2 - Electrokinetic sterility testing device

Info

Publication number: WO2007146677A2
Application number: PCT/US2007/070431
Authority: WO
Inventors: Daniel Armstrong
Original assignee: Daniel Armstrong
Priority date: 2006-06-06
Filing date: 2007-06-05
Publication date: 2007-12-21
Also published as: WO2007146677A3

Abstract

This invention relates to a small, hardy, less expensive, easy-to-operate electrokinetic instrument developed specifically for rapid sterility testing. The electrokinetic apparatus comprises a separation pathway; an electrical supply; two electric contact buffer pools; a sample solution inlet; a blocking agent reservoir; laser(s); and a photodetector. It utilizes a segment of dilute cationic agent to temporarily reverse the migration direction of the microbes, and another segment of solution containing a 'blocking agent' to hinder the microbe migration and focus them into a narrow zone.

Description

ELECTROKINETIC STERILITY TESTING DEVICE

FIELD OF THE INVENTION

This invention relates to a small, hardy, less expensive, easy-to-operate electrokinetic instrument developed specifically for the sterility testings.

BACKGROUND OF THE INVENTION

Some Research-grade electrokinetic devices, such as Capillary electrophoresis (CE), Electro-Chromatography (EC) instruments and chip-based microfluidic devices/instruments, can be adapted to do a sterility test. However, they are expensive, require an experienced operator/scientist and must be used properly and carefully operated to get the best results. In addition, since such research instruments are primarily used for the analysis of molecules and are engineered for high efficiency separation, there separation channels tend to be quit small in order to avoid the problem of Joule heating, which causes band broadening. It should be noted, that the small volume/diameter channels cause other unwanted effects for microbe separation. These include small sample volumes and short detection pathlength. Capillary electrophoresis (CE) devices are a family of related instruments that employ narrow-bore (20-200 mm i.d.) capillaries to perform high efficiency separations of both large and small molecules. These separations are facilitated by the use of high voltages, which may generate electroosmotic and electrophoretic flow of buffer solutions and ionic species, respectively, within the capillary. The properties of the separation and the ensuing electropherogram have characteristics resembling a cross between traditional polyacrylamide gel electrophoresis (PAGE) and modern high performance liquid chromatography (HPLC). CE is capable of routinely generating in excess of 500,000 plates per meter, much more than that is achievable by conventional HPLC. However, CE currently has limitations, for example the inability to resolve analytes with similar electrophoretic mobilities.

Electrochromatography device is a electrokinetic instrument using narrow bore packed column separations where the liquid mobile phase is driven not by hydraulic pressure as in HPLC but by electroosmosis. The advantages of using electroosmosis to propel liquids through a packed bed are the same as for in open capillaries i.e. reduced plate heights as a result of the plug flow profile and the ability to use smaller particles leading to higher peak efficiency than is possible in pressure driven systems (HPLC).

All electrokinetic instruments utilize two electrokinetic phenomena: electroosmotic flow (EOF) and electrophoretic movement. The driving force in electrokinetic methods is electroosmotic flow and the separation is due to the electrophoretic movement of the solutes. Electroosmotic flow results from the electrical double layer that exists at the liquid - solid interface between, in the case of CE, the bulk liquid and the capillary surface and in CEC Under alkaline conditions, the surface silanol groups of the fused silica or other approperiet media will become ionized leading to a negatively charged surface. This surface will have a layer of positively charged ions in close proximity which are relatively immobilized. This layer of ions is called the Stern layer. The potential at the boundary between the Stern layer and the interface with the diffused double layer, is called the zeta potential, and values range from 0-10OmV. Electroosmosis results because the core of liquid within this sheath will also be transported to the cathode. Electroosmotic flow depends upon the surface charge density, the field strength, the thickness of the electrical double layer, and the viscosity of the separation medium which in turn is dependent upon the temperature.

Solutes separate by virtue of their differences in electrophoretic mobilities. Electrophoretic mobility is the property of the movement of the charged species under the influence of an electric field. It results from the charge induced movement to the respective electrodes. Electrophoretic movement makes possible the simultaneous analysis of cations, anions, and neutral species in a single analysis. At neutral to alkaline pH, the EOF is sufficiently stronger than the electrophoretic migration such that all species are swept towards the negative electrode. The order of separation in this typical case is cations, neutrals, and anions. BRIEF SUMMARY OF THE INVENTION

This invention relates to an electrokinetic apparatus for rapid sterility testing including the parts of a separation pathway, an electrical supply, two electric contact pools, a sample solution inlet, a blocking agent reservoir, laser(s), and a photodetector. The electrokinetic apparatus further comprises a box with lid, read outs on the box, a control device, and an outlet to PC for control and read out.

Preferably, the electrokinetic apparatus in this invention has a separation pathway with a cross sectional area of 1800um to 250,000um . Preferably, the electrokinetic apparatus has the electrical supply with the voltage from IkV to 50KV.

The embodiment of the invention further provides the electric contact pools contain a running buffer with cationic surfactant in suitable concentration. It also provides that the electrokinetic apparatus has the blocking solution reservoirs containing a solution with a zwitterionic detergent in suitable concentration.

In some embodiments of this invention, the electrokinetic apparatus uses a UV detector. In some other embodiments, the electrokinetic apparatus utilizes a fluorescent detector. Preferably, the apparatus of this invention has the laser(s) set at the wavelength of 400nm -500nm, and the photodetoctor is with the wavelength of 550nm - 800nm. In some other embodiments, the electrokinetic apparatus has the laser(s) set at the wavelength of 550nni -800nm, and the photodetoctor is in the wavelength of near- IR range.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the basic setting of the apparatus of this invention.

Figure 2 illustrates the typical resulted electropherogram generated by connected PC, in which the first peak in the migration time is the detector's response to the EOF marker and the later sharp peak is the detector's response to the microbial band.

DETAILED DESCRIPTION OF THE INVENTION

There is needed a small, hardy, less expensive, easy-to-operate instrument that does just one thing, but does it very well and reliably: the sterility test. Such a machine may not be useful as a general, flexible, complex research instrument or for doing molecular separations. However, by simplifying the instrument, eliminating most of its research capabilities and substituting/utilizing hardy and enhanced components that are focused on its "sterility test" function.

The instrument uses electroosmotic force for pumping. It can be done by using a capillary, a channel, or a microfluidic chip (MFC) as the separation pathway. Referring to the figure 1 , the basic instrumental configuration for this invention is relatively simple. All that is required is a separation pathway with an optical viewing window which is aligned with the detecting system, a controllable high voltage power supply, two electrode connecting to two electric contact buffer pools, a electric contact blocking agent reservoir, and a sample solution reservoir or inlet, and a photodetecting system including lasers and photodetectors. The input end of the separation pathway is controlled by an input controller. After filling the capillary with buffer, the sample can be introduced by switching the end of the separation pathway into the sample solution or by hydrodynamic injection of the sample solution. Then the input controller switches to the blocking agent reservoir to introduce a segment of blocking agent solution to the pathway. After these introductions, the input end is switched back to the buffer pool and the separation continues.

The capillary used as a separation pathway may be coated. But the coating isn't to reduce the EOF, but for the ability to eliminate solute adsorption. The use of hydrophilic coatings can be useful in suppressing adsorption of hydrophobic compounds. Electrostatic binding can also be suppressed. Hydrophobic coatings, in conjunction with nonionic surfactants as buffer additives, appear promising as well.

The detector for this invention can be varied. Currently the vast majority of electrokinetic instruments in pharmaceutical analysis have involved the use of UV absorbance detection, including the use of indirect UV detection and low UV wavelength detection. New detection options on commercial instrumentation include diode array (DAD) and laser-induced fluorescence. Currently the use of laser-induced fluorescence (LIF) detection in this invention has to go with proper microbial fluorescent dying treatment of the samples. However, the additional steps of pre-separative sample derivatization are still in the scope of this invention.

Laser inducing fluorescence should be used for the LIF detection, but not with traditional, expensive lasers (like the Argon Ion laser). Small cheap diode lasers or other less expensive lasers will be used with special focusing lens. Multiple inexpensive lasers can be used to compensate for their lower power (if needed). Fluorescent light from any microbes present can be gathered (through a cut-off filter) into a fiber optic and carried to a photodetector. Alternatively the photodetector or photodetectors can be placed directly after the cut-off filter. Multiple detectors can be used to increase sensitivity if needed.

The sensitivity of using photodetector can improved by employing large sample volumes (i.e., long injection times) under stacking conditions. Using larger-bore capillaries than the standard can dramatically increase sensitivity. Capillaries used for typical CE instruments have an inner diameters range from 20-200 urn. From the standpoint of resolution, the smaller the capillary dimension, the better the separation. However, smaller-bore capillaries yield poorer limits of detection due to reduced detector path length and sample loadability. In this invention, the dimension of the pathway can be as high as 180,000 um², which will give a much better detection sensitivity. However, deleterious heating effects may occur and the buffer concentration, voltage, and pathway length should be adjusted to give an acceptable level of current. The dimensions of the pathway are larger than that for traditional CE, isoelectric focusing (IEF), isotachophoresis or capillary electro-chromatography. This is because a properly aggregated microbial sample will not be dispersed by Joule heating. Joule heating is a consequence of the resistance of the buffer to the flow of current. The practical voltage limit with today's technology is about 30 kV. By using higher voltages, and thus, it will provide greater efficiency by decreasing the separation time. Since narrow capillaries are also more prone to clogging, larger dimension pathway gives more tolerance for the particles in the buffer and sample solutions. Nevertheless, attention should be paid for sample preparation. It may be impractical to filter samples, therefore high speed centrifugation is usually used to settle suspended particles.

Operating Costs for this invention is relatively low. Typical settings employ aqueous electrolyte solutions with 10 to 20 mL being a common daily requirement. Costs are minimal when compared to HPLC solvent purchase and disposal. Uncoated capillaries may be generally employed which are a fraction of the cost of a HPLC column or specially coated capillaries.

This invention devises an appropriate electrokinetic system for sterility test. It allows the microbes, migrating as a single band, to distance themselves from the EOF, to distinguish them from contaminants appearing there. The general process to operate the instrument of this invention is set up in the following manner.

The capillary is rinsed with water and base and then filled with running buffer containing a cationic agent, such as CTAB in an appropriate concentration. The sample solution of microbes is then injected (it does not contain CTAB). Last, a segment of blocking agent, such as zwitterionic solution (which also does not contain CTAB) is placed in the capillary. The dissolved CTAB residing in front of the bacteria (on the anodic side) migrates toward the cathode when the voltage is applied. As it passes through the sample zone it carries the bacteria with it. The combination of the anodic movement of the blocking segment and the cathodic movement of the microbes allows them to converge at a point between the two zones in the capillary. The testing result can be illustrated as in the Figure 2, which is generated by the connected PC. In Figure 2, the microbial band migrates later than the EOF front and is detected as a sharp peak.

Without hydrodynamic sample injection, similar results may be generated by putting the end of the separation pathway into the electric contacted sample solution reservoir. The electroosmotic flow will draw the sample solution into the pathway.

One of the fundamental processes that drive this instrument is electroosmosis. The electroosmotic flow is reversed under the setting of this invention, and it flows toward the anode. For example, if we use the fused silica capillaries as the separation pathway, the fused silica surface has ionizable silanol groups in contact with the buffer contained within the capillary. The negatively-charged wall attracts positively-charged ions from the buffer. By using cationic surfactant in the running buffer, the surface of the capillary will be covered by a double layer of cationic surfactant, which reverses the surface charge to positive. This reversed positive charged inner surface attracts negatively charged ions from the buffer, creating an electrical layer. When a voltage is applied across the capillary, anions in the diffuse portion of the electrical layer migrate in the direction of the anode, carrying the bulk solution with them. The result is a net flow of buffer solution in the direction of the positive electrode. Under the setting of this electrokinetic device, the cationic agent(s), such as CTAB reverses the typical EOF to the direction of positively charged anode. However, CTAB agent itself will have a current motion counter to the electroosmotic flow and move into the region containing the microbes. When CTAB reaches the microbes section, it will interaction with the negatively charged microbes' membrane to reduce or stop their movement along with the EOF. Suitable cationic agents include cationic surfactants and geminal di-cationic liquid/salt.

A sharp peak will appear; when the microbes' counter current movement is hindered by a "blocking agent", which serves as a screen segment for the aggregated microbe particles to prevent them migrate out of the blocking region. The blocking agent is interactive with the aggregated microbe particles. Proper blocking agent can be a nutrient broth, a peptide, zwitterions, or zwitterionic detergent.

Along with the operation of the device, the microbes begin to aggregate and eventually form a large macroparticle. As the macroparticle forms, it quickly loses mobility, and from that point on it migrates with EOF in the anodic direction while residing in the blocking segment. The bacteria yielded peaks are typically have migration times longer than that of the EOF. The result may be generated by the connected PC as an electro-chromatographical chart as Figure 2.

The device of this invention provides a novel format for sterility testing which offers the following advantages:

• employs electrokinetic mechanism with great separation resolution;

• utilizes very high electric field strength for quick results; • uses larger dimension separation pathway for easy operation and better sensitivity;

• has time efficiencies higher than that of expensive instrument;

• requires minute amounts of sample;

• consumes limited quantities of reagents;

• is applicable to a wider selection of contaminated samples compared to other techniques.

Claims

Claims:

1. An electrokinetic apparatus for rapid sterility testing, comprises:

a separation pathway;

an electrical supply;

two electric contact buffer pools;

a sample solution inlet;

a blocking agent reservoir;

laser(s);

a photodetector.

2. An electrokinetic apparatus as claim 1, fiirther comprises:

a box with lid;

read outs on said box;

a control device on said box;

an outlet to PC for control and read out.

3. An electrokinetic apparatus as claim 1, where the separation pathway has a

cross sectional area of 1800um² to 250,000um².

4. An electrokinetic apparatus as claim 1 , where the electrical supply has the

voltage from IkV to 50KV.

5. An electrokinetic apparatus as claim 1, where the electric contact pools

containing a running buffer with cationic surfactant in suitable concentration.

6. An electrokinetic apparatus as claim 1 , where the blocking agent reservoirs containing a solution with a zwitteriomc detergent in suitable concentration.

7. An electrokinetic apparatus as claim 1 , where the photodetector is a UV detector.

8. An electrokinetic apparatus as claim 1 , where the photodetector is a fluorescence detector.

9. An electrokinetic apparatus as claim 1 , where the laser(s) set at the

wavelength of 400nm -500nm, and the photodetoctor is with the wavelength of550nm - 800πm.

10. An electrokinetic apparatus as claim 1, where the laser(s) set at the wavelength of 550nm -800nm, and the photodetoctor is in the wavelength of near-IR range.