WO2009017763A1

WO2009017763A1 - Multiple format biiosensor

Info

Publication number: WO2009017763A1
Application number: PCT/US2008/009215
Authority: WO
Inventors: John Lawarence Ervin; Hus Tigli
Original assignee: Trex Enterprises Corporation
Priority date: 2007-07-30
Filing date: 2008-07-30
Publication date: 2009-02-05
Also published as: EP2183351A1

Abstract

A multiple format label-free porous silicon based optical sensor. A first preferred embodiment provides a modular system that utilizes a micro-well based format, a single flow cell format and a multiple flow cell format. With each of these formats fluids containing bio-molecules within the pores of a porous silicon chip are optically analyzed to measure molecular interactions.

Description

MULTIPLE FORMAT BIOSENSOR

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of provisional patent applications Serial Nos. 60/962,652, 60/962,616, 60/962,664, 60/962,756, 60/962,675, 60/962,669 and 60/962,644 all filed 7/30/2007 and provisional patent application Serial No. 61/127,910, filed 5/15/2008 and is a continuation in part of Serial No. 1 1/180,349 filed 7/13/2005, Serial No. 10/631 ,592 filed 7/30/2003 and Serial No. 10/616,251 filed 7/8/2003.

FIELD OF INVENTION

This invention relates to optical biosensors and in particular to porous silicon biosensors.

BACKGROUND OF THE INVENTION

Optical Biosensors

An optical biosensor is an optical sensor that incorporates a biological sensing element. In recent years optical biosensors have become widely used for sensitive molecular binding measurements. To study interactions of proteins with other biomolecules one may generally use labeled or label-free methods. For these methods a first molecule of interest (the receptor) is immobilized onto a surface. An interaction is monitored by then introducing additional molecules (the targets) and detecting whether they in fact bind to the receptor. When using labels to monitor these interactions a fluorescent, colorimetric or some other signal is generated by an additional molecule or moiety that is attached to the target or receptor which gives a signal when the interaction takes place. This so called label (or tag) is present only to detect the interaction and is not part of the interaction of interest per se.

In label free binding, on the other hand, the receptor and target binding are monitored directly using untagged biomolecules. A variety of technologies exist in the art to detect binding without labels including surface plasmon resonance (SPR) and white light interferometery using porous silicon. In addition to the variety of technologies which exist to monitor label free binding events, there are a variety of instrument architectures which can used. These include plate readers and flow cells. In the case of plate readers a well plate (or micro well plate or micro titer plate) is used to house the biochips and fluids which are used for the label free binding studies. This allows for parallel analyses of several types of data. Alternatively flow cells house biochips in, typically, a microfluidic cell which routes fluid over the region of the biochip where the binding interaction takes place.

When acquiring and analyzing data of this sort there are a number of steps which are performed for the data analysis (the data method) on a number of channels (be those channels, flow cells or wells in a well plate). A file format which captures the full gamut of what a user of the analytical instrument might want to do must incorporate flexibility in acquisition and in analysis.

Surface Plasmon Resonance

An optical biosensor technique that has gained increasing importance over the last decade is the surface plasmon resonance (SPR) technique. This technique involves the measurement of light reflected into a narrow range of angles from a front side of a very thin metal film producing changes in an evanescent wave that penetrates the metal film. Ligands and analytes are located in the region of the evanescent wave on the backside of the metal film. Binding and disassociation actions between the ligands and analytes can be measured by monitoring the reflected light in real time. These SPR sensors are typically very expensive. As a result, the technique is impractical for many applications.

Resonant Mirror

Another optical biosensor is known as a resonant mirror system, also relies on changes in a penetrating evanescent wave. This system is similar to SPR and, like it, binding reactions between receptors and analytes in a region extremely close to the back side of a special mirror (referred to as a resonant mirror) can be analyzed by examining light reflected when a laser beam directed at the mirror is repeatedly swept through an arc of specific angles. Like SPR sensors, resonant mirror systems are expensive and impractical for many applications.

Separations-Based Measurements

More recently, optical biosensors have been used as an alternative to conventional separations-based instrumentation and other methods. Most separations-based techniques have typically included 1) liquid chromatography, flow-through techniques involving immobilization of capture molecules on packed beads that allow for the separation of target molecules from a solution and subsequent elution under different chemical or other conditions to enable detection; 2) electrophoresis, a separations technique in which molecules are detected based on their charge-to-mass ratio; and 3) immunoassays, separations based on the immune response of antigens to antibodies. These separations methods involve a variety of detection techniques, including ultraviolet absorbance, fluorescence and even mass spectrometry. The format also lends itself to measure of concentration and for non-quantitative on/off detection assays.

Thin Films

It is well known that monochromic light from a point source reflected from both surfaces of a film only a few wavelengths thick produces interference fringes and that white light reflected from a point source produces spectral patterns that depend on the direction of the incident light and the index of refraction of film material. (See "Optics" by Eugene Hecht and Alfred Zajac, pg. 295-309, Addison- Wesley, 1979.)

Kinetic Binding Measurements

Kinetic binding measurements involve the measurement of rates of association (molecular binding) and disassociation. Analyte molecules are introduced to ligand molecules producing binding and disassociation interactions between the analyte molecules and the ligand molecules. Association occurs at a characteristic rate [A][B]Ic_0n that depends on the strength of the binding interaction Ic_0n and the ligand topologies, as well as the concentrations [A] and [B] of the analyte molecules A and ligand molecules B, respectively. Binding events are usually followed by a disassociation event, occurring at a characteristic rate [A][B]Ic_0H- that also depends on the strength of the binding interaction. Measurements of rate constants Ic_0n and k_ofl- for specific molecular interactions are important for understanding detailed structures and functions of protein molecules. In addition to the optical biosensors discussed above, scientists perform kinetic binding measurements using other separations methods on solid surfaces combined with expensive detection methods (such as capillary liquid chromatography/mass spectrometry) or solution-phase assays. These methods suffer from disadvantages of cost, the need for expertise, imprecision and other factors.

Porous Silicon Layers

U.S. Patent No. 6,248,539 (incorporated herein by reference) discloses techniques for making porous silicon and an optical resonance technique that utilizes a very thin porous silicon layer within which binding reactions between ligands and analytes take place. The association and disassociation of molecular interactions affects the index of refraction within the thin porous silicon layer. Light reflected from the thin film produces interference patterns that can be monitored with a CCD detector array. The extent of binding can be determined from change in the spectral pattern. Prior art proposed techniques for utilizing porous silicon for optical analysis propose a micro-well format where porous silicon chips in wells of micro-well plates or they propose a format in which the chips are positioned in a flow cell through which the fluids containing the ligands and analytes flow.

Need What is needed is a multi-format biosensor.

SUMMARY OF THE INVENTION

The present invention provides a multiple format label-free porous silicon based optical sensor. A first preferred embodiment provides a modular system that utilizes a micro- well based format, a single flow cell format and a multiple flow cell format. With each of these formats fluids containing bio-molecules within the pores of a porous silicon chip are optically analyzed to measure molecular interactions.

A preferred embodiment is a multiple format label-free porous silicon based optical sensor having a base unit comprising a spectrometer based optical system comprising two light sources and two spectrometers. The system includes at least one single porous silicon flow cell unit, at least one multiple porous silicon flow cell unit and at least one micro- well plate adapted to hold a porous silicon chip in a plurality of micro wells. The system includes one or more exchangeable format trays adapted to position said single porous silicon flow cell unit, said multiple porous silicon cell unit and said micro-well plate serially within the base unit. The system also includes fluid systems adapted to provide fluids containing buffer solutions, ligand containing solutions, and analyte containing solutions to the single flow cell, said multiple flow cell unit and said micro-well plate. A control system with a computer processor provides automatic optical analysis of molecular interactions within the porous silicon chips in said single flow cell, said multiple flow cell or said micro-well plate depending on which format is at the time being utilized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. IA and IB show an example of a micro-well design with porous silicon chip at the bottom of the well.

FIG. 2 shows the general layout of a standard 96-well micro-well plate.

FIG. 3 is a drawing showing features of a molecular sensor system fabricated by Applicants and their fellow workers.

FIG. 4 is a flow cell diagram for the FIG. 3 system.

FIG. 5 shows temperature control elements and a triple format tray adapted for use of a single flow cell.

FIG. 6 is an outside view of a modular fludics unit for providing up to 6 different fluids to micro-well plates or flow cells.

FIG. 7 shows some of the internal features of the modular fluidics unit.

FIG. 8 shows storage cups for five buffer solutions.

FIG. 9 shows the triple format tray adapted for use of a 96-well micro-well plate.

FIG. 10 shows a 3-axis robotic unit for automatic loading of the 96-well micro-well plate.

FIGS. 1 1 AND 12 show elements of the optical components of the FIG. 3 system. FIG. 13 shows an X-Y robotic unit for automatic positioning of the optical beam on target regions.

FIG. 14 shows a blow-up of a portion of the flow cell shown in FIG. 5.

FIG. 15 is an enlargement of a portion of FIG. 14.

FIG. 16A shows an enlarged view of the gasket shown in FIG 3.

FIG. IB shows how fluid flows through a porous silicon chip and the region of the chip optically examined.

FIG. 17 shows a six flow-cell strip with a blow-up of the components of one cell. FIG. 18 show the fluid flow chart for a multiple flow cell format.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A preferred embodiment of the present invention is a porous silicon based optical biosensor similar to systems described in parent applications Serial No. 11/180,349 filed 7/13/2005, Serial No. 10/631,592 filed 7/30/2003 and Serial No. 10/616,251 filed 7/8/2003 and

Serial No. entitled "Optical Sensor and Methods for Measuring

Molecular Binding Interactions" which is being filed simultaneously with this application. All of the above applications are incorporated herein by reference.

First Preferred Embodiment Three-Format Molecular Sensor

A first preferred embodiment is a modular system providing three important formats for label-free study of protein interactions with porous silicon biochips uses. These are a micro-well based format, a single flow cell format and a multiple flow cell format. These three formats are summarized briefly below:

Micro-Well Based Format

In the micro-well format designed by Applicants, the porous silicon surface lies in the bottom of a standard micro-well to allow for integration into existing liquid handling and assay automation systems and optical monitoring from through the bottom of the micro- wells. In these units the fluid delivery is provided by an overhead liquid handling system or reagent pipettor. These units may be preferred to maximize assay throughput and to minimize costs per assay. These units can be in custom formats or can follow industry standard formats (e.g. formats established and maintained by the Society for Biomolecular Screening). This permits integration into other liquid handling and assay automation systems. Such formats include 96-well, 384-well and 1536-well layouts. A standard 96-well plate is shown in FIG. 3. These standard micro-well plates are available from many suppliers such as Corning, Inc.

Principle advantages of a micro-well format as compared to the flow cell format discussed above are as follows: elimination of micro-fiuidic channels and valves which reduces the costs of the assay disposables parallel processing of large numbers of samples which significantly improves assay throughput • compatibility with existing instrumentation and robotic equipment which permits integration with existing sample handling and preparation methods such as drug development libraries and clinical samples.

Binding experiments and binding assays in the micro-wells are conducted in a manner similar to that used in cartridge-based flow-systems described above except that a liquid handling system is used to transfer fluids and the fluids are not flowed across the biochips FIGS. IA and IB show cross-sections of micro-wells of micro-well porous silicon biosensor plates. FIG. IA shows a porous silicon surface 300 etched into silicon plate 302 forming the bottom 304 of a well 306. A fluid sample is shown at 308. In FIG. IB a porous silicon plate 310 is inserted at the bottom of a well made of any of a variety of materials. The fluid sample is also shown as 308. Molecular interactions take place within the pores of the porous silicon in each case which changes the index of refraction of the porous silicon layer. In preferred embodiments, as explained in detail elsewhere in this disclosure, broadband light from above reflected from the bottom and top of the porous silicon layer produces interference fringes which is monitored to determine characteristics of the molecular interactions.

Single Flow Cell Format

In the single flow cell format a single porous silicon chip is positioned within a removable cartridge that is positioned above a spectrometer based optical system. To study molecular binding interactions a micro-fluid system is provided that routes reference fluids and sample fluids across two portions of the single porous silicon chip. The optical system monitors in parallel through the bottom of the tray two portions of the chip, one portion is a region of the chip over which the sample fluids are flowing and the other portion is the region of the chip over which the reference fluids are flowing. The flow cell interfaces to the fluidics unit through the side by using a clamp type interface. A computer processor calculates a difference signal based the spectral information from the sample fluid to the reference fluid.

Multiple Flow Cell Format

In the multiple flow cell format up to 24 flow cells each with their own chip in strips of 6 flow cells. These are positioned within a removable plate that is positioned above a spectrometer based optical system. Like with the single unit, to study molecular binding interactions a micro-fluid system is provided that pumps reference fluids and sample fluids over two portions of a single porous silicon chip in each of the flow cells. The mechanics of the fluidic interface between the flow cell and the fluidics unit is also different than in the single flow cell setup. In this case a robot (called the AutoHandler) is used to bring the two inlets and outlets to the top of each of the flow cells. These flow cells are used in serially, one at a time. To start the acquisition at a particular flow cell, the optical head is brought directly below the flow cell and the AutoHandler robot is used to bring the fluid entry and exit directly above the flow cell where it makes a fluidic seal. After these two steps the flow cell currently measured is then used as a single flow cell instrument. The prior art requires user intervention when switching between several flow cells. Modular System

FIG. 3 is drawing of the complete three format molecular sensor system. It can be utilized for study of samples in standard 96-well micro-titer plates. It can be used to study kinetic reactions in a special flow cell designed by Applicants. And it can be used for study with multiple flow cells also designed by Applicants. This system consists of six modules: a base module 700, an autosampler 702, a fluidics unit 704, an autohandler 706, a buffer holder 708 and a computer processor 710 not shown. The modules are combined in ways which allow users to measure biomolecular interactions in flow cells or in well plates as well as allows for different levels of automation. The modules will be described in part and then as combined.

Base Module

The base module 700 includes an embedded controller, an optical subsystem, temperature subsystem, motion subsystem, and an exchangeable format tray. This module is present in all configurations of this embodiment. It is powered directly from wall plug AC power.

Controller

The embedded controller shown at 712 in FIG. 11 includes two circuit boards, a first board computer 711 running embedded linux and a second circuit board 713 used to communicate with other subsystems of the module. (The single board computer in this preferred embodiment may also be programmed in the graphical language Lab View.) The program running on the embedded computer contains a running server which is always passively awaiting commands before it reports status or performs any actions. Outside communication from the base is via a transmission control protocol (TCP/IP), and the base has two ports for this. One port is used to communicate with the controlling computer. The other port is used to communicate with the fluidics unit which also has an embedded controller of the same type.

Optical Subsystem

The base also consists of an optical subsystem used to monitor the binding event within the poSi biochips. This subsystem is mounted on the bottom of the top base plate and is shown in the FIG. 11 which is a view looking up at the bottom of this top plate. Important components of the optical subsystem is white light lamp 714 which is a quartz tungsten halogen lamp supplied by Ocean Optics with offices in Dunedin, Florida. Specifically this subsystem includes a visible white light source used to irradiate the poSi (though a single wavelength CW laser could also be used). In a preferred mode of operation, the light from the source is bifurcated into two fibers that are routed to a target test region of a porous silicon chip and a reference region of a porous silicon chip where it is focused to 200 μm diameter using a single lens. The light reflected from the reference portion and the test portion is then collected through the same optical fiber bundle and routed to separate spectrometers shown at 716 and 718 in FIG. 11 and 12.

FIG. 12 shows the optical fiber bundle routing used in the base module. Here the lamp is sent to an optical fiber bundle which is bifurcated into sample and reference arms. Each of these then goes though a single fiber in a one surrounded by six type bundle scheme (see inset). These 7 fibers are position at the poSi chip (one 7 fiber fiber bundle per poSi chip) with illumination being carried through the central fiber. The outer six fibers then collect the light and route it to the spectrometer where again there is one spectrometer for the sample and another spectrometer for the reference. The spectrometers are grating based spectrometers (Zeiss Model No. MMSl) with a 256 pixel linear NMOS array detector. The data from the spectrometers are then read out at fixed intervals using the single board computer and custom circuit board at 16 bits resolution. In this preferred mode of operation the processor reports difference information from the test region and the reference region virtually eliminating instrument noise. Also shown on FIG. 1 1 is 24 volt, 1 OA power supply 720.

The optical system also includes a motion subsystem 722 including an X-Y robot 724 used to raster the two channel probe head 726 of the optical subsystem as shown in FIG. 341. The head is moved so that the optical spots are directly underneath the region of the porous silicon chips to be interrogated. As stated above these chips could porous silicon chips in a flow cell or porous silicon chips in a well plate. This robot includes two direct current servo motors and under closed loop position control. Controllers are H-Bridge drive DPS controlled stand along controllers (Model MC-DC 3003 from Faulhaber) which are each controlled through a separate RS-232 communication link to the host processor.

Temperature Control

The temperature control system is used to control the temperature of the porous silicon biochips and is on board the base unit. In this preferred embodiment temperature can be controlled between 10.0 degrees 60.0 degrees centigrade in 0.1 degree increments. Components of the temperature control system are shown in FIG. 34B. These include four thermoelectric modules (not shown) in thermal contact with the exchangeable format tray 722 on one side and with heat sink fins 724 on the other. These thermoelectric units are driven by a 24V H-bridge circuit located at 726 as shown on FIG. 1 1 which provides up to 3 amperes of current to the units. The H-bridge is under analog control through the circuit board 713 shown in FIG. 11 which implements a closed loop proportional integral algorithm. Temperature monitoring is done on the H-Bridge board (for safety) via thermistors located on the exchangeable format tray 722 and the heat sink fins 724. When the heat sinks warm, fans (not shown) underneath them are turned on until the heat sinks return to the temperature required for accurate thermal control.

Exchangable Format Tray

Preferred embodiments of the present invention includes the exchangeable format tray 722 referred to above which is part of the base module and this exchangeable format tray holds the sample or samples (one or many flow cells or a micro-well plate) in a temperature controlled environment over the optical subsystem. The samples are optically monitored from the bottom. The tray permits a switch between a well plate format and a flow cell format in less than two minutes. Prior art optical sensors for molecular monitoring are dedicated to a single format such as: single flow cell, multiple flow cells or well plates, but not all of the above. The format tray is unique to the particular configuration but when changing between configurations only the format tray needs to changed. This anodized aluminum tray slides in place on the base unit and is held in place by spring plungers (not shown). This tray exists to accommodate a single flow cell, a sensor plate consisting of up to 24 flow cells (available in strips of 6) or a single, standard 96-well micro-titer plate.

Single Flow Cell

An exchangeable format tray adapted for single flow cell measurements in shown in FIG. 12 and 13 with the elements of a disposable cartridge shown as a blow-up. The disposable cartridge 760 contains a 7 X 3.5 mm porous silicon chip 762. Other elements of the disposable cartridge include cartridge housing 764 with a transparent bottom, compressor element 766, clamp 768, bottom gasket 770 and top gasket 772. FIG. 14 is an enlarged view of bottom gasket 770. Bottom gasket 9 is positioned under the porous silicon chip which sits above the gasket with its porous side against the bottom gasket.

FIG. 14B is a drawing of the porous silicon chip 762 looking at it through the bottom gasket 770 as the illumination elements does. Hole 774 in the gasket is provided to center it in relations to other components in the device. Sample fluid flows onto the porous silicon chip from the bottom through tubes 776 and 778 and reference fluid flows from the bottom through tubes 780 and 782. The fluid is trapped within the pores of the porous silicon chip 762 within regions 784 and 786 between the solid part of the porous silicon chip and a transparent bottom plate not shown. The porous silicon with the trapped fluid is illuminated from the bottom over region 788 for the sample and 790 for the reference. Reflections from two surfaces of the porous silicon are detected by the optical system to produce two interference patterns, one for the sample and one for the reference. Substantially these interference patterns are fit separately giving an OPD measurement for the sample and for the reference. The difference of these values is the differential signal which is used to characterize the extent of the biomolecular interaction. Applicants estimate that taking providing these differential patterns improves the resolution of these interference patterns by a factor of 8.

Multiple Flow Cells

The exchangeable format tray is adapted for multiple flow cells by having slots for up to four, six flow cell strips. A drawing of one six flow cell strip is shown in FIG. 34N with a blow-up of one of the cells. The tray has 48 holes in the bottom for the light to shine through. In the blow-up in FIG. 15 the porous silicon chip is shown at 792, a bottom gasket is shown at 794 and the inlet and outlet ports for the sample and the reference fluids is shown at 796. The flow cell strips, when placed down, have no fluidic interface to the fluidics unit. This interface is formed by the AutoHandler carrying two inlets and two outlets. A flow chart for the multiple cell format is shown in FIG. 18.

Micro- Well Plate

FIG. 9 shows the multiple format tray adapted for use with a standard 96-well micro-well plate. As shown in FIGS. IA and IB, porous silicon chips 300 are located at the bottom of each well and fluids are examined through the bottom of each well with the optical system as explained above.

Autosampler .

In this preferred embodiment fluid samples are provided automatically by a module that Applicants call their autosampler shown at 702 in FIG. 3. This unit is basically an off- the-shelf sampling unit used primarily in high performance liquid chromatography applications. The autosampler module is a customized version of the Alias Autosampler sold by Spark Holland in Emmen, Netherlands. This is a refrigerated two tray, closed frame autosampler that holds trays for either 48 vial trays, 96 well microtiter plates, or 384 well microtiter plates at between ambient temperature and 4 degrees Celsius. In chromatography application the autosampler is used with an internal valve for sample injections. Applicants have modified the unit to supply the sample loops of the fluidics unit. The autosampler has been fitted with a valve suitable for selection instead of one for injection. This allows a single autosampler to service three separate instruments for label free binding studies.

The autosampler has a movable tray holder, driven by a stepper motor based belt system which moves in the X direction. Aspiration is performed by a glass coated steel needle located on a gantry. The gantry is moved via a stepper motor driven lead screw in the Y- direction and by a stepper motor in the Z-direction. To address the several parts of the trays, the tray holder is moved in X, the gantry is moved in Y and the syringe goes down in Z the necessary amount. Aspiration and dispensing is performed with a stepper motor driven syringe pump connected to a 1 of three valve. In this valve the selection port is connected directly to the syringe, one of the ports is connected to a wash solvent bottle, one is connected to transfer tubing and the other is left unconnected.

The transfer tubing (which is two times the volume of the syringe needle) connects the syringe pump with a PEEK 1 of 6 selection valve internal to the autosampler. The incorporation of this valve is a customization not seen on the standard unit. This one of six valve has its selection port connected to the syringe pump via the transfer tubing has one port connected to the needle, one port connected to the sample injection valve on the Fluidics Unit, one port connected to the reference injection valve on the Fluidics Unit and the other three ports unused.

During a standard injection the syringe is routed to the proper tray position. The 1 of 3 valve on the syringe pump connects the syringe to the transfer tubing and the 1 of 6 valve connects the transfer tubing to the aspiration needle. The syringe pump then aspirates fluid through the needle and 1 of 6 valve to put fluid in the transfer tubing. The 1 of 6 valve then switches to route the sample in any direction needed (for instance to the sample or reference loops) and the syringe pump then switches to dispense mode.

The autosampler is controlled using a universal serial bus (USB) interfaces to the fluidics unit. The unit is powered directly from wall plug AC power.

Fluidics Unit

The fluidics unit is shown a 704 in FIG. 3. Flow paths are shown on FIG. 4. The unit contains an embedded controller, solvent delivery system and injection control and is mounted on the base unit to perform automated flow cell measurements. The unit is powered from 24V DC power available from the base unit or from the AC/DC converting power supply 720. The embedded controller 71 1 is a single board computer running embedded linux and a custom circuit board 713 used to send instructions to the solvent delivery system and injection control subsystems. The controller connects to the outside world through TCP/IP using a control program which acts as a server. The server is always running awaiting commands from a client which in the preferred embodiment is the base unit. The controller passes commands in the extensible markup language (xml) to the fluidics unit and the fluidics unit sends xml based responses back to the controller.

The control system in this preferred embodiment permits simultaneous measurement of a sample and reference channel in the flow cell modes. This differs from the prior art in which multiple flow cells are measured "in sequence" as opposed to "in parallel". In the preferred embodiment the fluidics unit 704 contains a continuous pump 738 operating at flow rates between 1 μL/min and 250 μL/min with 100 psi pressure capability. Pump 738 pulls buffer through a buffer selection system consisting of 5 vacuum degassing chambers 740. The buffer to be used is determined by the position on a six port selection valve integrated into the fluidics unit. Five of the six buffer storage tubes is shown in FIG. 34E at 741 ; the other is used without degassing. In a typical operation five storage tubes shown at 734 in FIG. 34E are loaded with sample fluids (which may include fluids such as water, pH 4.5 acetate, pH 7.5 phosphate buffered saline (PBS), 0.1 M ethanolamine, other aqueous buffers at a variety of pH and ionic strength, regeneraton solution such as H₃PO₄, and cleaning solutions like dilute bleach.

As shown at 742 in FIG. 4, the output of pump 738 is split into two equal flow paths using a passive y-configuration and sent to two, two position injection valves 744 and 746 each fitted with 50 μL volume sample loops contained in the fluidics unit. The above components are connected with 1/16" Teflon tubing from the buffers to the degassing chambers, from these to the selection valve, and from this to the passive Y. From the passive Y to the injection valves and to the flow cell the tubing is then 1/32" PEEKsil with 75 μm diameter as dispersion needs to be minimized between the injection valves and the flow cell. The fluidics unit is factory configurable with regard to numbers and types of pumps and valves. The description for the specific configuration exists on the fluidics unit in an xml form. This configuration is passed to the controlling computer which then draws a picture of the fluidics system on a graphical user interface (GUI). This picture is then used to graphically control the instrument through the GUI. Having the physical hardware described as xml permits the hardware to be controlled by many different software programs giving unparalleled flexibility in the realm of label-free interaction analysis.

AutoHandler

While the autosampler module picks samples (answering the question: What should be used?) the autohandler robot, shown at 706 in FIG. 3, routes these samples or fluids to the appropriate place for analysis (answering the question: Where should it go?) The addition of this second robot in the system permits the system to handle multiple flow cells and multiple wells in a plate. The addition of the autohandler permits complete, partial, or no automation depending on the needs for the system. This preferred embodiment permits up to 24 flow cells (available in modules of six) to be placed at the instrument at the same time and addressed individually. The autohandler is powered from 24V from the base unit for from a AC/DC transforming power supply.

The autohandler interfaces with the base unit through a USB connection. The autohandler robot is shown separately in FIG. 34G and contains four stepper motors and motor controllers. These form an X,Y,Z,Θ motion system. The X-rail is shown at 750; the Y-rail is shown at 752 and the Z lead screw is shown at 754. A dispensing syringe needle is shown at 756. The X and Y motors align to the flow cell or well in X and Y and then lowers the gantry in the Z direction. The theta movement switches between the fluidic interface and a simple dispensing needle. All motors on the autohandler are used in closed loop mode.

Buffer Holder

A buffer holder connects to the base through a single USB connection and is powered through that connection. This holder is used to hold and monitor the buffers used for the interaction analysis. The buffer level is monitored through weight. The buffer holder, holds up to 6 buffers each stored in 50 mL centrifuge tubes. Buffer monitoring ensures that when long evaluations are started, that there is enough buffer to complete the run.

Control Computer

In this preferred embodiment a control computer handles method programming, data acquisition and data analysis for the instrument system. This computer exists on the same network as the base instrument and communicates with the base module. In this preferred embodiment the control computer always interacts to everything via the base module through a single TCP/IP connection.

Other Preferred Embodiments

The three-format system of the first preferred embodiment provides an extremely versatile optical sensor. There are many variations that should be obvious to persons skilled in this art based on the disclosures provided above. For example some cost savings would result from not providing for the multi-flow cell format or the micro-well format. Also various configurations are possible with the first preferred embodiment as described below:

Configurations

This preferred embodiment may be operated in a large number of configurations including: manual plate reader, fully automated plate reader, partially automated plate reader, manual single flow cell, automated single flow cell and automated multiple flow cells. These configurations are summarized below:

Manual Plate Reader Configuration

For the manual plate reader only the base and client computer are needed. In this case the well plate tray is used and fluids are introduced manually by pipetting. Here the motion subsystem of the base is used to raster the fiber optic bundles under the proper well plate to be addressed. The biochips are held into place using injection molded polycarbonate well strips which are strips of eight that fit into the microtiter plates. These well strips are manually placed into the well strip as needed.

Fully Automated Plate Reader Configuration

For an automated plate reader solution, the manual plate reader configuration is augmented by the autosampler and autohandler. In this case the autosampler is used to aspirate and dispense samples as appropriate. It answers the question what is needed. The autohandler then routes these samples to the appropriate well. It answers the question, where do the samples go. The autohandler is also used to pick and place the well strips used to hold the biochips. In this way for a binding experiment, the prepared chips are submerged in fluid that sets the initial time = 0 of the experiment.

Partially Automated Plate Reader Configuration

In this case the base, client computer and autohandler are used. For this instrument configuration, the user uses offline equipment to perform the liquid handling necessary to prepare a 96 well plate with see through bottom. The prepared plate is put on the instrument and biochips are introduced into the appropriate wells by the autohandler. As before readout is from the bottom of the plate using the motion subsystem on the base.

Manual Single Flow Cell Configuration

For the partially automated flow cell, the base (using the flow cartridge exchangeable plate), fluidics unit and client computer are used. Here a user will manually fill the injection loops on the fluidics unit. By following a program setup in the client computer, the fluidics unit will then proceed to perform the binding experiment while the base reads out the data through the bottom of the flow cell. In this case the motion subsystem of the base unit is not necessary to move. The buffer storage unit may also be added to this configuration. In this case buffers levels are monitored in real time.

Automated Single Flow Cell Configuration

For the fully automated single flow cell configuration the base the autosampler is added to the manual single flow cell configuration. Here instead of manually filling and washing the sample loops of the fluidics unit, the autosampler will do this. In this case many samples may be entered into the autosampler and run sequentially in an unattended manner. The buffer storage unit may also be added to this configuration.

Automated Multiple Flow Cell Configuration

In this case the base is fitted with the multiple flow cell tray that holds either 6, 12, 18 or 24 differential flow cells - to form a so called sensro plate. To this base is added the autosampler, the autohandler, the fluidics unit and the client computer. The autohandler has the role of forming a fluidics connection to the several flow cells as they are used. In this case the X and Y axes of the autohandler are used to align to the proper position on the sensor plate. The Z-axis brings the fluidic connection down to the sensor plate. As before with the Automated Single Flow Cell configuration, the autosampler is used to load and wash the sample loops on the Fluidics Module. The fluidics unit pumps the fluid as needed and handles buffer changes and sample loading and injection.

The motion subsystem of the base unit is used to bring the optical probe under the flow cell currently being addressed. In this way, after instrument setup, the instrument can take a large amount of data on many systems sequentially. The buffer storage unit may also be added to this configuration.

Variations

While there have been shown what are presently considered to be preferred embodiments of the present invention, it will be apparent to those skilled in the art that various other changes and modifications can be made herein without departing from the scope and spirit of the invention. Therefore, the scope of the invention should be determined by the appended claims and their legal equivalents and not by the examples that have been given.

Claims

What is claimed is:

1. A multiple format label-free porous silicon based optical sensor providing three formats of operation, i.e., a single flow cell format, a multiple flow cell format and a micro-well format, said system comprising:

A) a base unit comprising a spectrometer based optical system comprising at least one light source and two spectrometers,

B) a single porous silicon flow cell unit,

C) a multiple porous silicon flow cell unit,

D) a micro-well plate adapted to hold a porous silicon chip in a plurality of micro wells,

E) one or more exchangeable format trays adapted to position said single porous silicon flow cell unit, said multiple porous silicon cell unit and said micro-well plate serially within said base unit,

F) a plurality of fluid systems adapted to provide fluids containing buffer solutions, ligand containing solutions, and analyte containing solutions to said single flow cell, said multiple flow cell unit and said micro-well plate,

G) a control system comprising a computer processor adapted to provide automatic optical analysis serially of molecular interactions within the porous silicon chips in said single flow cell, said multiple flow cell or said micro-well plate, depending on which of the three formats is being.

2. The sensor as in Claim 1 wherein said system is configured for manual plate reading.

3. The sensor as in Claim 1 wherein said system is configured for fully automated plate reading.

4. The sensor as in Claim 1 wherein said system is configured for partially automated plate reading.

5. The sensor as in Claim 1 wherein said system is configured for manual single flow cell operation.

6. The sensor as in Claim 1 wherein said system is configured for automated single flow operation.

7. The sensor as in Claim 1 wherein said system is configured for automated multiple flow cell operation.