HIGH SENSITIVITY REAL-TIME BACTERIAL MONITOR

Abstract

Systems for the monitoring of bacterial levels in samples, using spectral analysis of the light diffracted from a substrate with an ordered array of pores having diameters enabling the targets to enter them. The trapping pore array is cyclically illuminated by light of different wavelengths, and the light diffracted from the pore array is imaged by a 2-dimensional detector array, with one pixel, or a small group of pixels receiving light from each associated pore. The temporal sequence of frames provides a series of images, each from the reflection of a different wavelength. A time sequenced readout of the signal from the pixel or pixels associated with each pore region, provides a spectral plot of the reflected light from that pore region. Spectral analysis of the light intensity from this series of different wavelength enables the effective optical thickness (EOT) of each pore to be extracted.

Claims

1. A method for detecting target elements in a host analyte, said method comprising: providing a substrate containing an ordered array of pores formed in its surface, at least some of said pores having lateral dimensions enabling said target elements to fit therein; directing said host analyte over the surface of said substrate, such that at least one of said target elements can enter at least one of said pores; Illuminated the pore array by light containing different wavelengths; imaging light diffracted from the pore array by a two dimensional detector array, with a pixel or a group of pixels receiving light from an associated pore; generating a temporal sequence of frames, each from the diffraction of a different wavelength; generating a time sequenced readout of the signal from said pixel or group of pixels associated with said pore region; and spectrally analyzing said time sequenced readout to enable the effective optical thickness of said associated pore to be determined.

2. A method according to claim 1 wherein said temporal sequence of frames, each from the diffraction of a different wavelength is generated either by filtering said illuminating light to generate a sequence of illuminating wavelengths, or by filtering said light diffracted from said pore array to generate a sequence of detected wavelengths.

3. A method according to claim 2 wherein said filtering is performed either by a filter wheel or a tunable liquid crystal filter.

4. A method according to claim 1, wherein said effective optical thickness of said associated pore is determined by performing a Fourier transform on said time sequenced readout of the signal from said pixel or group of pixels associated with said pore.

5. A method according to claim 1, wherein said host analyte including said target elements is directed, prior to being directed over said surface of said substrate, in a stream in contact with a filter membrane which enables said host analyte to pass therethrough but not said target elements, such that the concentration of said target elements in said stream increases with passage of said stream towards the surface of said substrate.

6. A method according to claim 1, wherein said host analyte including said target elements is directed towards said surface of said substrate in a direction such that it impinges said surface at an angle substantially different from a grazing angle.

7. A method according to claim 1, wherein said pores are provided with a material which preferentially attracts said target elements, said material being disposed in said pores remotely from the surface of said substrate.

8. A method according to claim 1, further comprising the application of an electric field generally perpendicular to the plane of said substrate, such that any target elements carrying a charge are preferentially attracted to enter said pores.

9. A method according to claim 1, wherein said target elements are bacteria.

10. A system for detecting target elements in a host analyte, said system comprising: a substrate containing an ordered array of pores formed in its surface, at least some of said pores having lateral dimensions enabling said target elements to fit therein; a flow system for directing said host analyte over the surface of said substrate; an Illuminating source generating light containing different wavelengths, said source being disposed such that said substrate is located in the light path of said illumination; a two dimensional pixelated array imaging light diffracted from said substrate, a pixel or a group of pixels receiving light from an associated pore; a tunable filter disposed in said light path between said illuminating source and said detector array, said filter generating a temporal sequence of wavelengths from said illumination; a frame recording system, configured to generate a temporal sequence of image frames of said pixelated array, each frame being recorded at a different wavelength; a spectral analyzer configured to extract from said temporal sequence of image frames, a spectral signal sequence from said pixel or group of pixels receiving light from said associated pore; and a processor adapted to determine from said spectral signal sequence, a measure of the effective optical thickness of that pore associated with said pixel or group of pixels.

11. A system according to claim 10 wherein said tunable filter generating a temporal sequence of wavelengths from said illumination, is disposed either in the path of light incident on said substrate, or in the path of light diffracted from said substrate.

12. A system according to claim 11 wherein said filter is either a filter wheel or a tunable liquid crystal filter.

13. A system according to claim 10, wherein said effective optical thickness of said associated pore is determined by performing a Fourier transform on said spectral signal sequence of the signal from said pixel or group of pixels associated with said pore.

14. A system according to claim 10, wherein said host analyte including said target elements is directed, prior to being directed over said surface of said substrate in a stream in contact with a filter membrane which enables said host analyte to pass therethrough but not said target elements, such that the concentration of said target elements in said stream increases with passage of said stream towards the surface of said substrate.

15. A system according to claim 10, wherein said host analyte including said target elements is directed towards said surface of said substrate in a direction such that it impinges said surface at an angle substantially different from a grazing angle.

15. A system according to claim 10, wherein said pores comprise a material which preferentially attracts said target elements, said material being disposed in said pores remotely from the surface of said substrate.

17. A system according to claim 10, further comprising electrodes for the application of an electric field generally perpendicular to the plane of said substrate, such that any target elements carrying a charge are preferentially attracted to enter said pores.

18. A method according to claim 10, wherein said target elements are bacteria.

19. A method for detecting target elements in a host analyte, said method comprising: providing a substrate containing an ordered array of pores formed in its surface, at least some of said pores having lateral dimensions enabling said target elements to fit therein; directing said host analyte including said target elements over the surface of said substrate, such that at least one of said target elements can enter at least one of said pores; illuminating at least part of said substrate with light; detecting illumination diffracted from said substrate with a two dimensional pixelled array, to provide a temporally sequenced, spectrally coded series of frames, each frame showing said illumination diffracted from said substrate at sequential time intervals; generating from said temporally sequenced series of frames, a reflected spectrum signal sequence for separate pixels or predetermined groups of pixels of said array; and determining from said reflected spectrum signal sequences, a measure of the effective optical depth of those pores associated with said separate pixels or predetermined groups of pixels, wherein said spectral coding is generated either by filtering said light to generate a sequence of illuminating wavelengths, or by filtering said detected illumination to generate a sequence of detected wavelengths.

20. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The presently claimed invention will be understood and appreciated more fully from the following detailed description, taken in conjunction with the drawings in which:

[0034] FIG. 1 shows a schematic illustration of a prior art system as shown in International Publication number WO 2014/155381;

[0035] FIG. 2 illustrates how the probability of impact of the bacterial content on the pore array is increased by aligning the flow direction to impinge at an angle on the surface of the pore array;

[0036] FIG. 3 illustrates a further implementation for creating motion of the bacteria in the preferred direction toward the pores by use of a nutrient layer at the bottom of the pores;

[0037] FIG. 4 illustrates schematically a device using a filter membrane to increase the probability of trapping bacteria in the pores, by increasing the effective concentration of bacteria in the analyte fluid flowing across the entrance of the pores;

[0038] FIG. 5 is a schematic illustration of a complete optical detection system for bacterial monitoring using a sequentially scanned wavelength source for illuminating the sample; and

[0039] FIG. 6 is a schematic drawing showing how the analysis of the temporally changing reflected interference light from the pixels of the array can be used in order to determine the EOT of individual pores, and hence the bacterial concentration of the solution being monitored; and

[0040] FIG. 7 illustrates a compact implementation of the bacterial monitoring system shown in FIG. 5.

DETAILED DESCRIPTION

[0041] Reference is now made to FIG. 1, which illustrates schematically a prior art system such as that described in International Publication number WO 2014/155381. Details of the structure of the system have been given in the Background Section of this disclosure. Optically, a broadband white light source illuminates the array, and the zero order reflected light is collected from a wide area of the array, and directed from the detector element 26 to a spectrometer. The spectrometer converts the light to a set of electrical signals proportional to the intensity of each wavelength included in the incoming light. The detected light is a combination of the reflected light from all the pores under the beam. The percentage of pores occupied by bacteria changes the intensity of the EOT signal proportionally to the total number of pores under the beam. It is necessary to wait until at least a few percent of the pores change their reflection pattern due to bacteria population, before a distinctive change in the EOT is noticed.

[0042] Reference is now made to FIG. 2, which illustrates how, according to a first exemplary arrangement, the probability of impact of the bacterial content on the pore array is increased by aligning the flow direction 22 to impinge on the surface of the pore array at an angle far from the glancing angle used in the prior art implementation of FIG. 1, such that the probability of bacterial entry into the pores is increased. In the implementation of FIG. 2, the entry of a bacterium into a pore may be achieved not only by means of its own motility, but also by the flow direction 22 of the analyte forcing it to impinge on and enter into a pore. The flow should be slow enough that the solution current within the pores will remain moderate to allow the bacteria to attach to the walls of the pores before they could be swept out of the pores again by the force of the flow. The velocity inside the pore should be less than 30 /sec, which is the order of the bacteria self-motion velocity.

[0043] Reference is now made to FIG. 3, which illustrates a further implementation for creating motion of the bacteria in the preferred direction toward the pores. It is known that the motility of bacteria in non-neutral solutions can be used to encourage movement of the bacteria towards or away from parts of the solution with higher concentrations of certain chemicals. This motion is known as Chemotaxing. For example, in the case of a gradient of concentration of bacterial nutrient, which is the material which the bacteria use as food, the bacteria motion will not be completely random in all direction, but the bacteria will prefer to move toward the higher concentration of nutrients. In the case of material with poisoning effects on the bacteria, a motion with preferred direction away from the high concentration is observed.

[0044] FIG. 3 illustrates a method by which this effect can be used to create preferred motion of the bacteria toward the array, by creating a high concentration of nutrients in the pores 23. At the bottom of the pores a layer of nutrient material is provided such as by making the pores extend through their bottom end, and positioning a layer of nutrient 30 beneath the substrate 20, as shown in FIG. 3. The nutrient layer 30 may either be positioned at the bottom of simple single diameter pores, or a stepped pore structure may be used, with micro-channels 31 from the bottom of the pores 23 to the bottom of the array substrate 20. This can be readily achieved by means of an additional etching step to the pore etching process. Positioning the pore array substrate 20 on top of an additional substrate composed of nutrient material 30, will cause the nutrients to diffuse 32 from the nutrient substrate into the pores, making the pores nutrient rich. The bacteria in the solution above the array will have now the motivation to move toward the array and into the pores. The probability P(I) for bacteria to move towards the array is now higher than the probability that they move away from the array, and in addition, the length of the leg of linear motion before a bacterium changes direction is also substantially longer.

[0045] Reference is now made to FIG. 4, which illustrates schematically a further implementation for increasing the probability of trapping bacteria in the pores, by increasing the effective concentration of bacteria in the analyte fluid flowing across the entrance of the pores. In the analysis cell of FIG. 4, a special filtration system is used in order to direct the host analyte with an increased concentration of the bacteria in the direction where it will impinge on the top of the pore array, while allowing the host solution itself (referred to as the permeate) to preferentially move in another direction, avoiding passage over the substrate, such that the bacteria content of the analyte moving towards the substrate (referred to as the Retentate) increases as the analyte itself is filtered out of the flow towards the substrate.

[0046] Referring to FIG. 4, the solution 40 enters through an inlet port 41 on the left-hand side of the drawing of the device, passes across the surface of the MPSiAS pored substrate 20, and exits the device through and outlet port 42 on the right-hand side of the drawing of the device. The inlet flow is directed down a passage 43 towards the pored substrate. The passage 43 may be tapered such that the narrowing of the passage causes an increased pressure of the solution. The envelope of the passage 43, through which the solution is passing, comprises a filter membrane which allows molecules of the solution to pass through 44, but blocks the passage of bacteria, which continue downwards towards the MPSiAS 20. As a result, the Primate solution tends to short-circuit 47 the path through the device from input to output port, flowing around the central opening 45 through which the optical illumination and detection is performed, and in so doing, increases the bacterial concentration of the Retentate solution which continues to flow down the input passage 43 and across the surface of the pored substrate 20. When finally arriving at the pored array 20, the bacteria concentration in the solution may be orders of magnitude higher than that of the solution 40 entering the device, thereby increasing the probability of bacteria entering the pores by the same concentration ratio increase.

[0047] A numerical example shows this effect quantitatively. Assuming that the height of the cell above the array is 0.1 mm, and the array width is 10 mm, the cross section of flow above the array is 1 mm.sup.2. If the entrance is designed such that the cross section of the solution flowing through the input port is 100 mm.sup.2, then using the proper membrane design (pore size and surface area), the bacterial concentration can increase by two orders of magnitudes before reaching the array, thereby increasing the detection sensitivity of the device by the same two orders of magnitude.

[0048] Reference is now made to FIG. 5, which is a schematic illustration of the complete optical detection system for bacterial monitoring of a host solution. The system differs from that shown in the WO 2014/155381 prior art, primarily in that the white light, broadband illumination source beam 50 generated in the illumination source 25 is passed through a rotating filter wheel 51, or any other tunable filter device for generating a sequentially changing wavelength beam. The sequentially variable wavelength beam may be directed towards the array 20 through a beam splitter 52. The zero order reflected light, from all the pores, is directed back through the beam splitter and imaged on a CCD array 53, which can be a black-and-white detector array. The frame received on the CCD is now a 2D map of the interference reflection of each point on the array, at the selected wavelength. The CCD used may conveniently be a widely available conventional array, having 1 or more Megapixels, and having a refresh rate of a low few tens of Hz, though it is to be understood that these values are not meant to limit the scope of the invention, but merely represent currently readily available, low-cost technology. Storing the frame in a frame stacker 54 and repeating the operation with each new wavelength, generates a temporal set of frames of 2D images of the reflection from the array, each one at a different wavelength, as indicted by the sample frame images 55, 56, 57, each representing the 2-D array image at successive time intervals, a different illuminating wavelength being present at each successive time interval. Processing the images and collecting the reflection of each pixel at a different wavelength, generates a 3D array of spectra of all the pixels, two dimensions being of the 2-D array of pixels in each frame, and the third dimension being the time scale over which the sequence of frames are stored in the frame stacker 54.

[0049] Since each pixel has collected the spectrum from a single point of the array, the change of the EOT generated from that specific pixel represent changes at the respective pore region of the array. It is possible to design the optics such that it will represent a small group of pores, down to one pore per pixel. Capturing of one bacterium in one pore now generates a large change in the collective reflectance from a region of just a few pixels and even 100% in the case of one pore per pixel. It should be noted that the pore size for commonly monitored bacteria are of the same order of magnitude as the pixel spacing of commonly available CCD or CMOS imaging arrays, a standard pixel being about 7 7 , while a suitable pore size for such bacteria is about 2 4 , and the space between the pores is also 3 5 . Consequently, it is possible to insert magnifying optics in the imaging path, and to either image one pore on one or several pixels or to have one pixel imaging several pores.

[0050] In an embodiment where the illuminating beam incident on the substrate is white light, and the spectral decoding is performed by means of a filter positioned in the reflected path of the illumination light, the filter wheel 51, or an alternative tunable filter device, could be positioned in front of the CCD detector array 53, instead of at the output of the illumination source 25, as shown in the implementation of FIG. 5.

[0051] Reference is now made to FIG. 6, which is a schematic drawing showing how the analysis of the temporally changing reflected interference light from the pixels of the 2D imaging array can be used in order to determine the EOT of individual pores, and hence the bacterial concentration of the solution being monitored. The light 61 reflected from a pored substrate 20 impinges on the pixels of a 2-D imaging array such as a CCD. In order to explain the information extraction procedure, the pixels are marked with indices according to their column and row, as follows: 11, 12, 13, . . . ; 21, 22, 23, . . . ; 31, 32, 33, . . . ; etc. The time sequenced output of each pixel is represented by an array of light intensity measured by that pixel as a function of time, as shown by the assembly 62 of intensity/time graphs, each graph representing the time dependent output of a single pixel of the array, as follows: I11, I12, I13, . . . ; I21, I22, I23, . . . ; and so on for the whole array. Each of those time dependent plots thus represents the spectral output of each pixel, since the wavelength detected is a direct function of the time elapsed in each wavelength scan cycle. Therefore, if each time sequenced output frame is collected in a frame stacker, the individual pixel intensity/time profiles, which represent the spectral intensity output from each pore region, can be analyzed, to provide the EOT of the pore region imaged by each pixel, as shown by the assembly 63 of EOTs, as follows: EOT11, EOT12, EOT13, EOT21, EOT22, EOT23, and so on.

[0052] The result of the cyclically sequentially changing wavelength illumination beam thus results in a series of reflected zero order interference signals, one from each pixel, each having the same temporally changing wavelength sequence as the input illumination. The interference spectrum is now given for each pixel, in the form of a time varying spectral trace, and by storing the signal outputs from each separate pixel sequentially, a spectral trace is obtained for each pixel of the interference pattern from the incident illumination. The number of sequential frames stored to generate a single spectral trace is synchronized with the rotation of the filter wheel, or with the wavelength cycle used if another type of time-varying filter is used. Spectral analysis of the temporal traces from each pixel, such as by Fourier transformation, enables the values of the EOT for each pixel to be obtained separately.

[0053] The difference between this method and that of the WO 2014/155381 prior art is that whereas in the prior art, the spectral analysis is performed repeatedly at a single instance of time for light collected from the entire pore array, and from the entire light source spectrum, in the present apparatus, the spectral analysis is performed on a spectrum accumulated from a temporally selected sequence of frames over the cycle of time of the varying input illumination wavelength. The resulting output representing the changes of the EOT's of each of the pixels of the 2D image, is now an indication of the changes of the occupancy of the pore or group of pores imaged on that specific pixel. A single event of a bacterium captured in one of the pores will be clearly delineated due to its impact on the reflection of that specific pore. One measurement of the combined EOT change of large number of pores is now replaced with large number of measurements of EOT's changes of single pores. As a result, it now becomes possible to detect bacterial concentrations less than 100 cells/ml, as opposed to the 10.sup.6 cells\ml of the prior art system.

[0054] Since the frame rate of currently used common CCD arrays is of the order of 30 Hz, and the processing of each pixel can thus be completed in several msec., a complete wavelength plot with typically 30 different measurement wavelengths can be completed within 1 sec. In practice, signal to noise limitations do not enable a useful signal analysis to be performed on a single wavelength cycle of typically one second, and accumulation times of one minute or so are required to obtain signal levels sufficiently good to make reliable bacterial concentration measurements down to 100 cells/ml.

[0055] Finally, reference is now made to FIG. 7, which illustrates a particularly compact implementation of the bacterial monitoring system shown in FIG. 5. In this system, the optical components are assembled on top of the flow cell 70, such that the dimensions of the monitoring system are minimized. The light source 71 projects its beam through a liquid crystal tunable filter 72, this being a very compact tunable filter having no moving parts, and from there through a beam splitter 73 onto the flow cell 70. The spectrally coded light reflected from the pores within the flow cell 70 are directed by the beam splitter 73 through an imaging lens arrangement 74 onto the CCD detector array 75. All of these optical components can be conveniently mounted in a small volume on top of the flow cell itself. Finally an electric field can be applied across the flow cell, such as by installing electrodes on the top and bottom surfaces 76, 77, of the flow cell, in order to enhance the probability of the charged bacteria from entering the pores of the substrate.

[0056] It is appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove. Rather the scope of the present invention includes both combinations and subcombinations of various features described hereinabove as well as variations and modifications thereto which would occur to a person of skill in the art upon reading the above description and which are not in the prior art.