WO2015161219A1

WO2015161219A1 - Microdialysis platform

Info

Publication number: WO2015161219A1
Application number: PCT/US2015/026414
Authority: WO
Inventors: Champak Das; Anu AGGARWAL; Brad LEDDEN; Guochun WANG; Sai Kumar; Qian Sun; Chien Nguyen
Original assignee: SFC Fluidics, Inc.
Priority date: 2014-04-17
Filing date: 2015-04-17
Publication date: 2015-10-22

Abstract

A multi-analyte detection platform for microdialysis introduces dialysate fluid and one or more reagents into a detection assembly and analyzes the dialysate fluid in near real-time for small or large molecules. The platform uses individually operable actuators to introduce the dialysate fluid and one or more reagents into individual wells of a well plate. An optical assembly detects the color generated from a reaction between the dialysate fluid and one or more reagents. An integrated electrical board performs the functions of the analysis and an onboard monitor provides a user the ability to control the platform through a graphical user interface. The platform is capable of detecting the concentration of different molecules in the dialysate fluid.

Description

MICRODIALYSIS PLATFORM

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims priority from U.S. provisional patent application number 61/980,983, filed on April 17, 2014, and entitled "Portable Cerebral

Microdialysis Platform." Such application is incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Phase-I and

Phase-ll funds from grant no. 1 R43NS076167-01 and 2R44NS076167-02A1 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] The field of the invention is microdialysis, and in particular to an automated microdialysis platform for near real-time analysis.

[0004] Microdialysis is a well-known technique of sampling biomolecules by means of an implantable probe consisting of a membrane and an appropriate perfusion mechanism. It is widely used in neuroscience research since its inception almost three decades ago. Microdialysis is an organ-specific technique, depending on its physiological location; its potential application has been demonstrated in the liver, skin, blood, stomach, ears, eye, and brain. This technique, notwithstanding its extensive use in research, has not realized successful transition to actual clinical use, mainly because of the logistical inconvenience and the questionable clinical value of the information obtained from a patient care perspective. The current method of monitoring based on microdialysis does not involve an all-in-one system; rather, the dialysate samples are collected periodically, labeled and stored, and subsequently analyzed offline in batches using bulky instruments. This protocol not only requires trained practitioners, but also is prone to errors in handling tiny amounts of dialysate. The significant delay in the reported data may render this data clinically irrelevant, inappropriate, or unusable. Moreover, current commercial instruments are limited to the analysis of small molecule metabolic biomarkers like lactate, glutamate, pyruvate, and glucose; these instruments do not detect large molecule biomarkers such as proteins. Availability of a nurse-friendly, fully integrated system that automatically collects, analyzes, and reports the dynamic changes in concentrations of clinically relevant biomarkers in near real time could significantly ameliorate the limitations of microdialysis for patient care in clinical applications, such as Neurointensive Care Units (NICU).

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to an automated and integrated bedside microdialysis platform with automated protocols for moving the perfusate from a reservoir to the microdialysis probe, and collecting and transporting the dialysate from the probe to an analyzer module wherein, in certain implementations, both small and large molecule biomarkers such as lactate, pyruvate, glutamate, glucose and S100B are detected and can be reported continuously for up to 24 hours without user intervention. A disposable dialysate cartridge precludes cross-contamination issues. This platform is applicable, in certain implementations, for early detection of biochemical changes that lead to secondary and comorbid pathologies after moderate to severe traumatic brain injury (TBI). The early biomarkers of these biochemical changes are best tracked with cerebral microdialysis, and continuous online monitoring at bedside provides meaningful medical intervention in clinical settings such as Level 1 trauma centers. Other implementations may be used in conjunction with different types of probes that are designed for detection of biomarkers in other bodily fluids, including but not limited to blood and urine.

[0006] These and other features, objects and advantages of the present invention will become better understood from a consideration of the following detailed description of the preferred embodiments and appended claims in conjunction with the drawing as described following:

BRIEF DESCRIPTION OF THE DRAWING

[0007] Figs. 1A and 1 B show an integrated X-Y slide with a well plate holder.

[0008] Fig. 2 is an assembly drawing showing the dispensing station consisting of independent multiple Z-direction actuators with fluid delivery lines.

[0009] Fig. 3 is drawing showing the analyte detection assembly using an optical detection assembly.

[0010] Fig. 4 shows the storage cartridge for reagents with options for

temperature control.

[001 1 ] Fig. 5 shows the assembly of the X-Y slide, dispensing station with Z- actuators, analyte detection assembly and reagent cartridge storage system.

[0012] Fig. 6 shows the assembly of the X-Y slide, dispensing station with Z- actuators, analyte detection assembly, reagent cartridge storage system, and additional peristaltic pumps.

[0013] Fig. 7 is a drawing of the complete microdialysis platform.

[0014] Fig. 8 is a schematic drawing of the entire system.

[0015] Fig.9 is a schematic drawing of the reagent filling station and detection assembly.

[0016] Fig. 10 is a schematic drawing of the actuation arm operation for

reagent/sample delivery for small molecule detection with larger proteins.

[0017] Fig. 1 1 is a schematic drawing of the actuation arm operation for

reagent/sample delivery for small molecules.

[0018] Fig. 12 is a graph showing the detection of Lactate in the dialysate in the instrument.

[0019] Fig. 13 is a graph showing the detection of S100B (protein) in the

dialysate in the instrument.

[0020] Fig. 14 is a graph showing the detection of glutamate and lactate (small molecules) in dialysate using electrochemical sensing.

[0021 ] Fig. 15 shows one configuration of the dispensing station with multiple Z- actuators.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S) [0022] In certain implementations, the invention is directed to a microdialysis platform that provides a method of analyzing the dialysate for both small and large molecules in a real-time, continuous manner. The platform is low maintenance and only requires user intervention for reagent/plate placement, which is required only every eight to twelve hours or, in some implementations, up to twenty-four hours. There are currently no known microdialysis instruments capable of the simultaneous real-time analysis of many different biomarkers of both small and large molecules in the same platform.

[0023] The microdialysis platform according to certain implementations is capable of working in combination with a dialysate collection unit, whereby a constant stream of dialysate is fed into the invention from said dialysate collection unit. The invention, however, is not limited to working in combination with a dialysate collection unit, and in certain implementations can work without the dialysate collection unit provided the invention is supplied with dialysate for analysis.

[0024] Figure 7 shows a diagram of the complete and assembled microdialysis platform 10, showing the analyte housing for microdialysis platform 10, while a schematic for the overall operation of the microdialysis platform is shown in Fig. 8. The microdialysis platform receives dialysate, which may come from a dialysate collection system or some other provider, which is carried out in one of a 96-well plate or 384-well plate or a similar high density well plate (for example, a 1536-well plate). The microdialysis platform has the option for either an optical or electrochemical method of detection. In this implementation using a probe designed for detection of biomarkers in cerebrospinal fluid, the microdialysis platform is capable of detecting a variety of molecular analytes, including lactate, pyruvate, glucose, glutamate, urea, and S100B protein. In one implementation, the system performs a continuous detection of small molecules (such as lactate, pyruvate, glucose, and glutamate), and in another implementation, the microdialysis platform adds an additional ability to detect S100B protein or any other proteins which can be detected by immunoassay. Other implementations may use different types of probes to detect biomarkers in, for example, extracellular fluid, blood, urine, or other bodily fluids.

[0025] For the analysis of small molecules in cerebrospinal fluid, enzymatic

reaction with color generation is carried out in individual wells with a certain amount of dialysate and two parts assay reagents, which are dispensed into the wells via a dispensing station. Figure 1 B shows an X-Y slide 12 with a well plate 14 (in this example, the plate has 384 wells) mounted in that system, while Figure 1A shows well plate 14 being placed in or removed from X-Y slide 12. The X-Y slide 12 is automated and can be programed to move laterally in an X- direction and/or Y-direction so that individual wells of the X-Y slide can be analyzed, as a specific position will correspond to a specific step of the analysis process. For example, one position will be associated with reagent loading, while another will be associated with detection, as shown in Figure 9. The lateral movement of the X-Y slide 12 allows each individual well of well plate 14 to move to each specific position associated with a specific functionality.

[0026] The reagents and dialysates are introduced to the individual wells of well plate 14 via a reagent and dialysate dispensing station 16, as shown in Figure 2. The dispensing station 16 comprises multiple individually addressable z-axis actuators 20 which move the associated delivery fluidic line 18 down to the individual wells of the well plate 14. In certain implementations, different wells are used to detect different molecular analytes, and thus multiple Z-direction actuators 20 in dispensing station 16 are employed for reagents along with one for the dialysate, although only one actuator 20 is shown in Figure 2 for clarity.

[0027] Although the configurations may vary, one such configuration would detect four small molecules (such as lactate, glutamate, glucose, and pyruvate) from the dialysate simultaneously, as shown in Figure 1 1 . In this configuration, four individually addressable actuators 20 are configured to dispensing station 16. The first actuator 20 (Z actuator-1 ) contains fluid delivery lines 18 for twelve individual wells in a three-by-four pattern. These delivery lines comprise standards for each of the four small molecules. For each analyte there are three standards so that a dynamic calibration curve can be generated, against which the sample will be compared to report its concentration in real-time.

[0028] For a microdialysis platform using optical detection, a second actuator 20 (Z actuator-2) contains fluidic lines 18 for delivering a Reagent A and a Reagent B for both lactate and pyruvate. A third actuator 20 (Z actuator-3) contains fluidic lines for delivering reagent for both glucose and glutamate. In a system using electrochemical detection, the third actuator 20 (Z actuator-3) contains

electrochemical sensors 22 for measuring glucose and glutamate, as shown in Figure 1 1 . Some examples of commercially available electrochemical sensors (EC sensors) include sensors from Pinnacle Technology, Inc. and Sarrisa

Biomedical. These electrochemical sensors are used to detect electrical signal from a reaction between a dialysate fluid and at least one reagent. The fourth actuator 20 (Z actuator-4) connects to an arm that carries the sample dialysate delivery line. [0029] In a second configuration, depicted in Figure 10, additional protein detection immunoassay is integrated with the small molecule detection described previously. This configuration uses three additional actuators 20 to provide a delivery mechanism for secondary antibody and to provide a wash line. Further modification may be possible to have a single line of glucose and glutamate with electrochemical sensors 22 for electrochemical detection. Chemi-luminescence and fluorescence detection may also be employed in various implementations. Figure 15 shows one configuration of the dispensing station 16, this configuration consisting of multiple z-actuator arms 20, each of which may have a unique function as described herein. The invention may also use a hybrid approach, whereby some of the molecules are detected by colorimetric detection and some others are detected by electrochemical sensing, or a hybrid of other forms of detection including chemi-luminescence and fluorescence detection. By integrating different techniques in one platform, it helps in reducing size, complexity and improving sensitivity and robustness in some cases.

[0030] In certain configurations, once the sample dialysate is delivered into

individual wells in addition to the reagents for enzymatic assay, the resultant reaction generates color, which can be detected with an integrated absorption based optical detection system. The relative position of the optical detection assembly with respect to the X-Y slide 12 is shown in Figure 3. The optical detection assembly comprises a light source assembly 24, including a filter, steering optics, and collimation optics, and a detector 26. The detector 26 may be a photomultiplier tube, a Photodiode, a CCD camera, or a CMOS based camera in various implementations. Because the X-Y slide 12 is capable of moving laterally in an X-direction and/or Y-direction, each individual well of well plate 14 can be moved inside the optical detection assembly's detection area.

[0031 ] The reagents used for this invention are stored in a reagent cartridge 28, which may be fitted with a temperature control unit, as shown in Figure 4. The temperature of the reagents must be kept at a constant temperature for the period of operation, such as eight to twelve hours of operation in certain implementations, in order to ensure optical performance and accurate reporting of concentration. Figure 5 shows the reagent cartridge inserted into the analyte housing, which is shown assembled with the dispensing station and analyte detection assembly on platform base. Figure 6 shows the reagent cartridge inserted into the analyte housing, which is shown assembled with the dispensing station and analyte detection assembly, which is further connected to peristaltic pumps 15 which, in one configuration, may be used to provide dialysate fluids to the detection assembly.

[0032] The microdialysis platform comprises an on-board electronic control board and software for performing all necessary protocol and final data analysis. An on-board screen or monitor consists of a graphical user interface for operating the invention. This microdialysis platform detects specific concentrations (relevant to clinical range) of different molecules in the presence of all the interferents present in the interstitial fluid. Figure 12 shows the calibration curve for lactate detection from dialysate performed in the instrument as an example. Similarly S100B detection in dialysate performed in the instrument is shown in Figure 13. Figure 14 shows detection of lactate and glutamate in electrochemical sensing mode in dialysate.

The present invention has been described with reference to certain preferred and alternative embodiments that are intended to be exemplary only and not limiting to the full scope of the present invention, as set forth in the appended claims.

Claims

A microdialysis platform for detecting the concentration of one or more analytes, comprising:

a. an analyte housing;

b. a dispensing station mounted to the analyte housing;

c. a reagent cartridge removably insertable into the analyte

housing;

d. an analyte detection assembly within the analyte housing; e. an electronic control board in communication with the

dispensing station, reagent cartridge, and analyte detection assembly, and configured to perform protocol and data analysis and

f. a monitor in communication with the electronic control board and configured to display a graphical user interface.

The microdialysis platform of claim 1 , wherein said analyte housing comprises an X-Y slide, wherein said X-Y slide can move in an x- direction and a y-direction.

The microdialysis platform of claim 2, wherein said analyte housing further comprises a well plate mounted to the X-Y slide, wherein said well plate comprises a plurality of individual wells.

The microdialysis platform of claim 3, wherein said electronic control board is configured to move the X-Y slide in the x-direction and the y-direction to position any particular one of the individual wells of the well plate into a specific detection position.

5. The microdialysis platform of claim 3, wherein the well plate

comprises a 96-well plate.

6. The microdialysis platform of claim 3, wherein the well plate

comprises a 384-well plate.

7. The microdialysis platform of claim 3, wherein the well plate

comprises a 1536-well plate.

8. The microdialysis platform of claim 1 , wherein the dispensing

station comprises a plurality of z-direction individually operable actuators.

9. The microdialysis platform of claim 8, wherein the reagent cartridge comprises a reagent temperature control unit.

10. The microdialysis platform of claim 9, wherein at least one of the individually operable actuators is connected to the reagent cartridge by a reagent fluidic line to deliver a reagent to at least one well of the well plate.

1 1 . The microdialysis platform of claim 10, wherein at least one of the individually operable actuators is connected to a dialysate source by a dialysate fluidic line to deliver a dialysate to at least one well of the well plate.

12. The microdialysis platform of claim 9, wherein said reagent

temperature control unit is configured to maintain reagents within the reagent cartridge at a constant temperature.

13. The microdialysis platform of claim 8, wherein the dispensing station comprises four individually operable z-axis actuators, wherein a first of said individually operable actuators connects to a standard delivery fluidic line for delivery to at least one analyte standard to at least one well of the well plate, further wherein a second of said individually operable actuators connects to a reagent fluidic line for delivery of at least one reagent to at least one well of the well plate, further wherein a third of said individually operable actuators is connected to an electrochemical sensor, and further wherein a fourth of said individually operable actuators is connected to a dialysate delivery fluidic line for delivery of a dialysate to at least one well of the well plate.

14. The microdialysis platform of claim 13, wherein the reagent

dispensing station further comprises three additional individually operable actuators, wherein a first of said three additional individually operable actuators is connected to a first antibody delivery fluidic line for delivery of at least one first antibody to at least one well of the well plate, further wherein a second of said three additional individually operable actuators is connected to a second antibody delivery fluidic line for delivery of at least one second antibody to at least one well of the well plate, and further wherein a third of said three additional individually operable actuators is connected to a wash line to deliver a wash fluid to at least one well of the well plate.

15. The microdialysis platform of claim 8, wherein the dispensing

station comprises four individually operable actuator assemblies, wherein a first of said individually operable actuator assemblies comprises at least one analyte standard fluid delivery line, further wherein a second of said individually operable actuator assemblies comprises at least one reagent delivery line, further wherein a third of said individually operable actuator assemblies comprises a reagent delivery line, and further wherein a fourth operable actuator assembly comprises a dialysate fluid delivery line.

16. The microdialysis platform of claim 1 , wherein the analyte detection assembly comprises an optical detection assembly to detect a color generated from a reaction between a dialysate fluid and at least one reagent.

17. The microdialysis platform of claim 16, wherein said reaction

between a dialysate fluid and at least one reagent is a chemi- luminescence reaction.

18. The microdialysis platform of claim 16, wherein said reaction

between a dialysate fluid and at least one reagent is a fluorescence reaction.

19. The microdialysis platform of claim 16, wherein the optical detection assembly comprises a light source, a filter, at least one steering optical element, at least one collimation optical element, and a detector.

20. The microdialysis platform of claim 19, wherein the detector is a photomultiplier tube.

21 . The microdialysis platform of claim 19, wherein the detector is a photodiode.

22. The microdialysis platform of claim 19, wherein the detector is a CCD camera.

23. The microdialysis platform of claim 19, wherein the detector is a CMOS based camera.

24. The microdialysis platform of claim 1 , wherein the analyte detection assembly comprises an electrochemical detection assembly to detect electrical signal from a reaction between a dialysate fluid and at least one reagent.

25. The microdialysis platform of claim 1 , further comprising a dialysate providing assembly to deliver a dialysate to the analyte detection assembly, wherein the dialysate providing assembly comprises a peristaltic pump and a dialysate collection unit.

26. The microdialysis platform of claim 1 , wherein the analyte detection assembly is configured to detect one or more of lactate, pyruvate, urea, S100b, glucose, and glutamate.

27. The microdialysis platform of claim 1 , wherein the analyte detection assembly is configured to detect any small or large molecule.

28. The microdialysis platform of claim 1 , further comprising a probe in fluid communication with the analyte detection assembly.

The microdialysis platform of claim 28, wherein the probe is configured to detect at least one biomarker in a bodily fluid selected from the group consisting of cerebrospinal fluid, extracellular fluid, blood, and urine.