WO2022250988A1

WO2022250988A1 - System and methods for detection of compounds and metabolites

Info

Publication number: WO2022250988A1
Application number: PCT/US2022/029293
Authority: WO
Inventors: Oshiorenoya AGABI; Renaud RENAULT
Original assignee: Koniku Inc.
Priority date: 2021-05-24
Filing date: 2022-05-13
Publication date: 2022-12-01

Abstract

A testing method for detecting a compound or a metabolite includes collecting saliva from a subject's mouth by inserting an absorbent section of a swab into the mouth. The absorbent section of the swab is then inserted into a biochip containing living cells. Fluorescence from the living cells is detected. A compound or metabolite in the saliva is identified based on the reaction of the living cells. The method may include sliding the absorbent section over a first side of a membrane in the biochip wherein the living cells are on a second side of the membrane. Biochips, swabs and kits are adapted for performing the methods.

Description

SYSTEM AND METHODS FOR DETECTION OF COMPOUNDS AND METABOLITES

TECHNICAL FIELD

[0001] The field of the invention is detection of compounds and metabolites. BACKGROUND OF THE INVENTION

[0002] Volatile organic compounds (VOCs) are natural or manmade compounds which readily diffuse into air, due to their volatile characteristics. Many VOCs are toxic to humans and the environment with extended exposure. VOCs are also associated with explosives. Thus, detecting VOCs is important to human safety and security, and for better preserving the environment. Although various techniques have been proposed and used for detecting VOCs, they have been met with only varying degrees of success. Accordingly, improved systems and methods for detecting VOCs are needed.

[0003] Human saliva contains compounds or metabolites indicative of various disease and health conditions, and call detect the presence of drugs, alcohol and other substances. Analyzing human saliva can therefore be useful in several ways.

BRIEF STATEMENT

[0004] The inventors have now developed a system for detecting VOC’s using living biological cells. From an evolutionary perspective, biological cells as a system have been fine-tuned over millions of years for the purpose of sensing various molecules. Cells have evolved to be energetically efficient and sturdy. Cells can repair themselves and adapt to environmental changes. Cells can also be reprogrammed and manipulated in a variety of ways through genetic modifications.

[0005] In humans, the sense of smell is generally achieved by a type of neuron located in the nasal epithelium, which express olfactory receptors (OR) on their surfaces. Each olfactory neuron usually expresses only one OR gene among the hundreds present in the organism’s genome. When an odorant molecule, or VOC, from inhaled air binds to a matching receptor, the event triggers a chain of reactions that result in electrical signals. These signals, or spikes, propagate into the brain and are further processed to give rise to a complex sense of smell. [0006] OR activation eventually results in an increase in cytosolic calcium concentration, which can be measured using a calcium sensitive fluorescent reporter such as GCaMP6f. The binding of an odorant molecule to its receptor induces an increase in the fluorescence emitted by the cells. An optical detector can therefore be used to measure cellular response in a contactless manner. The present system and methods detect VOC’s using an optical detector that detects fluorescence.

[0007] The inventors have also developed systems and methods for detecting compounds and metabolites in saliva or other liquids using cells with specific receptors.

[0008] Other objects, features and advantages will become apparent from the following detailed description and drawings, which are provided as examples for explanation, and are not intended to be limits on the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In the drawings, the same element number indicates the same element in each of the views.

[0010] Fig. 1 is a schematic diagram of a VOC detection system.

[0011] Fig. 2 is a schematic diagram of the optical system of the VOC detection system of Fig. 1.

[0012] Fig. 3 is a bottom perspective view of a microfluidic biochip.

[0013] Fig. 4 is a top perspective view of the microfluidic biochip shown in Fig. 3.

[0014] Fig. 5 is a bottom perspective view of the microfluidic biochip of Figs. 3 and 4 with the top foil or seal layer shown in Fig. 4 removed for purpose of illustration.

[0015] Fig. 6 is an exploded top perspective view of the microfluidic biochip shown in Figs. 3 and 4.

[0016] Fig. 7 is a schematic representation of an osmolarity control system.

[0017] Fig. 8 is a front perspective view of a detection system with the top cover removed for purpose of illustration. [0018] Fig. 9 is a side perspective view of the detection system of Fig. 8 with the top cover in place.

[0019] Fig. 10 is an enlarged front view of components of the detection system shown in Figs. 8 and 9.

[0020] Fig. 11 is a front view of components of the detection system shown removed from the housing.

[0021] Fig. 12 is a top view of the optical system having four optical channels, for use in the detection system shown in Fig. 1.

[0022] Fig. 13 is a front view of a biochip loader.

[0023] Fig. 14 is a side view of the biochip loader shown in Fig. 13.

[0024] Fig. 15 is a top view of the biochip loader shown in Figs. 13 and 14.

[0025] Fig. 16 is a top view of the biochip loader of Figs. 13-15 positioned for loading and unloading biochips from the detection system shown in Figs. 8-12.

[0026] Fig. 17 is another front perspective view of the detection system of Fig. 8.

[0027] Fig. 18 is a top view of a biochip for use with saliva or other liquid.

[0028] Fig. 19 is a top view of a swab for use with the biochip shown in Fig. 18.

[0029] Fig. 20 is a top view illustrating insertion of the swab of Fig. 19 into the biochip of Fig. 18.

[0030] Fig. 21 is an exploded perspective view of the biochip shown in Figs. 18 and 20.

[0031] Fig. 22 is a top view of the swab of Fig. 19 in its original state before use.

[0032] Fig. 23 is a top view of the swab of Figs. 19 and 22 showing a color change after saliva is applied to the sponge section of the swab.

[0033] Fig. 24 is a side view of the biochip and swab as shown in Fig. 18.

[0034] Fig. 25 is a side view of the swab of Fig. 23 partially inserted into the biochip of Figs. 18 and 21. [0035] Fig. 26 is a side view of the swab of Fig. 23 fully inserted into the biochip of Figs. 18 and 21.

[0036] Fig. 27 is a section view of a locking mechanism in the biochip of Figs. 18 and 21.

[0037] Fig. 28 is a top view of another biochip for use with saliva or other liquid.

[0038] Fig. 29 is a top view of a swab for use with the biochip shown in Fig. 28.

[0039] Fig. 30 is a top view illustrating insertion of the swab of Fig. 29 into the biochip of Fig. 28.

[0040] Fig. 31 is an exploded side view of the biochip shown in Figs. 28 and 30.

[0041] Fig. 32 is a top view of the swab of Fig. 29 in its original state before use.

[0042] Fig. 33 is a top view of the swab of Figs. 29 showing a color change after saliva is applied to the sponge section of the swab.

[0043] Fig. 34 is a side view of the biochip and swab as shown in Fig. 30.

[0044] Fig. 35 is a side view of the swab of Fig. 33 inserted into the biochip of Fig. 31.

[0045] Fig. 36 is a top view of another biochip for use with saliva or other liquid.

[0046] Fig. 37 is a top view of a swab for use with the biochip shown in Fig. 36.

[0047] Fig. 38 is a top view illustrating insertion of the swab of Fig. 37 into the biochip of Fig. 36.

[0048] Fig. 39 is an exploded perspective view of the biochip shown in Fig. 36.

[0049] Fig. 40 is a top view of the swab of Fig. 37 in its original state before use.

[0050] Fig. 41 is a top view of the swab of Fig. 37 showing a color change after saliva is applied to the sponge section of the swab.

[0051] Fig. 42 is an exploded perspective view of the biochip shown in Fig. 36 and the swab as shown in Fig. 37. [0052] Fig. 43 is an exploded perspective view of the swab of Fig. 37 inserted into the biochip shown in Fig. 36.

[0053] Fig. 44 is a section view of a locking mechanism in the biochip of Fig. 36 and 43.

[0054] Fig. 45. is a side view of a detection system and a used biochip collection container.

[0055] Fig. 46 is a perspective view of the collection container shown in Fig. 45. DETAILED DESCRIPTION

[0056] Referring to Figs. 1 and 2, in a basic form, a VOC detection system 20 includes a cell carrier or substrate, such as a microfluidic biochip 22, an optical system 24 and an electronic system 26. The microfluidic biochip 22 contains cells 30, medium or water 32, and a membrane 36 which provides a barrier for the cells against contaminants such as viruses, bacteria and dust. The cells bind to the membrane 36, allowing the cells to more effectively interact with airborne odorants such as VOC’s. Each channel or optical pathway of the optical system 24 includes one or more: light emitter, such as a blue LED 46, lenses 40A, 40B 40C and 40D, optical filters 42A and 42B, dichroic mirror 44, and a photodetector such as a photodiode 48.

[0057] Fig. 1 shows an embodiment having two optical pathways each having the above-listed elements, although the system may be designed with a single optical pathway or multiple optical pathways, depending on the intended application. The electronic system 26 in Fig. 1 is electrically connected to the blue LEDs 46 and to the photodiodes 48 and may include a digital lock-in amplifier 52 in the form of a field programmable gate array (FPGA). The electronic system 26 has an output device, such as a thin film transistor (TFT) display. Alternatively, the output or reporting of from the detection system 20 may be provided via a WIFI, cellular, RF or wired connection. The electronic system 26 may include a GPS unit for detecting and reporting the location of the detection system 20. The electronic system 26 may also include control software or circuitry, and memory for recording detection events and other data. The detection system 20 may be powered by a battery 28, to allow flexibility in placement and use. [0058] Turning now to Figs. 3-6, in the example shown, specifically in Fig. 6, the microfluidic biochip 22 has a bottom or first layer 68, a second layer 66, a third layer 64, a fourth layer 62, and a fifth or top layer 60. The layers may be laser cut from PET plastic sheets (polyethylene terephthalate). The layers may be attached and sealed together via an adhesive, or by using bio-compatible double sided tape and a hot press. The layers may optionally be made of glass and/or PDMS (silicon-based organic polymer) assembled using plasma bonding. The layers below the cells are transparent, so that the cells may be exposed to a light source such as the blue LED 46, and so that fluorescence emitted by the cells may be detected by the photodiodes 48. The layers above the cells 30 may optionally be transparent so the cells may be viewed from above.

[0059] As shown in Figs. 5 and 6, the fifth layer 60 and the fourth layer 62 have through holes providing wells 72 for holding cells 30. A membrane 65, such as a PTFE membrane, on the bottom surface of the third layer 64 closes off the bottom of the wells 72. The membrane may be treated to make it transparent and to promote cell adhesion. Cell adhesion to the membrane allows for better detection of VOCs, which move from the air flow channel 84 in the biochip 22 through the membrane 65. Although the example shown has four wells 72 in a square array, other numbers, patterns and shapes of wells may be used. Capillaries 80 in the fourth layer 62 connects a water inlet 76 in the fifth layer 60 into each of the wells 72. The capillaries 80 may be etched into the fourth layer 62 before assembly.

[0060] An air inlet 74 extends through the fifth layer 60, the fourth layer 62, the third layer 64 and connects into the air flow channel 84 which is formed in the second layer 66. As shown in Fig. 6 the air flow channel 84 extends under each of the wells 72, in an S- shaped configuration. The membrane 65 encloses the air flow channel 84 from above while the first layer 68 encloses the air flow channel 84 from below. The membrane 65 separates the cells 30 in the wells 72 from the air flow channel 84. The air flow channel 84 may be wider at positions under the wells 72, so that the cells 30 are better exposed to elements such as VOCs moving through the membrane 65.

[0061] Fig. 6 shows the second layer 66 attached to the first layer 68 and to the third layer 64 using layers of double sided tape 66A and 66C, as one example. The layers may alternatively be attached using adhesives, fasteners, plastics welding, or other techniques. Alignment holes 82 may be provided at the corners of each layer to precisely align the layers on a fixture during assembly of the layers into the microfluidic biochip 22. After the microfluidic biochip 22 is assembled and ready for use, cells 30 are placed into the wells 72 from the top of the fifth layer, the cells are seeded on top of the membrane 65, and the cells bind to the membrane 65. A foil or seal layer 70 may then be adhered onto the top surface of the fifth layer 60 to cover and seal the wells 72, as well as the water inlet 76, the air outlet 78, and the air inlet 74. The foil or seal layer 70 also prevents light from entering the top of biochip 22. This reduces evaporation and avoids stray light affecting the signal from the photodetectors. The microfluidic biochip 22 is then effectively sealed against the environment. The biochip 22 may be manufactured as a disposable unit intended for replacement e.g., every 30 days.

[0062] The microfluidic biochip 22 is designed for operation in the detection system 20 shown in Figs. 1 , 2 and 8-10, although it may also be used in other systems as well. Referring to Figs. 8, 9, 10 and 17, in the detection system 20, the optical system 24, the electronic system 26 and the battery 28 are contained within a housing 90. A frame 112 is positioned on top of the base 110. The frame 112 has a detection system slot or front opening 136 adapted to receive the microfluidic biochip 22. The base 110 and the frame 112 may be fixed in position on guide posts 128. A top plate 114 is supported on one or more jack screws 120 which are rotated by one or more jack screw motors 122. The jack screws 120 and the jack screw motors 122 form an elevator to raise and lower the top plate 114 towards and away from the frame 112. Bushings at the corners of the top plate 114 slide on the guide posts 128 and prevent lateral movement as the top plate 114 moves vertically. Alternatively, the top plate 114 may be fixed in position with the frame 112 and the microfluidic biochip 22 moved vertically.

[0063] A water or medium supply container 94 is connected to a water supply tube 96 which passes through the top plate 114, at a position in alignment over the water inlet 76 of the microfluidic biochip 22, when the microfluidic biochip 22 is installed in the detection system slot 136. A vacuum tube 100 extends from a water collection container 104, through the top plate 114, to a position aligned over the air outlet 78 of the microfluidic biochip 22 when the microfluidic biochip 22 is installed in the detection system slot 136. An air inlet tube similarly extends through the top plate 114 to a position aligned over the air inlet 74 of the biochip 22. With the top plate 114 in the up position, the biochip is sealed from the environment. When the top plate 114 moves down to engage the biochip 22, the water supply tube 96, the vacuum tube 100 and the air inlet tube pierce through the seal layer 70 to make fluid connections with the biochip 22.

[0064] A pump tube 102 connects the inlet of a vacuum pump 98 to the water collection container 104. The outlet of the vacuum pump 98 leads to an outlet port 108. In an alternate design, a positive pressure pump may be used instead of the vacuum pump 98, with air pumped into the air inlet and through the air flow channel under positive pressure, rather than drawing air through the air flow channel via vacuum.

[0065] The detection system components may be in or on a housing 90 enclosed by a cover 92. As shown in Fig. 9, sight windows 106 may be provided through side walls of the housing 90 aligned with the water supply container 94 and the water collection container 104, to allow visual inspection of the water level in the containers. The detection system 20 does not actively remove water from the biochip 22. However, humidity in the air moving through the air flow channel may condense into liquid water, which moves into and is collected in the water collection container 104.

[0066] As shown in Fig. 8, the outlet port 108 may extend through a front wall of the housing. Also as shown in Fig. 8, the electronic system 26 may include an on/off switch 132 on the housing 90, and a USB port 134 for charging the battery 28 or for interfacing the electronic system 26 to another device via a USB cable. As shown in Figs. 10 -12, rollers 126 projecting into the detection system slot 136 are rotatable to guide the biochip 22 into the detection system slot 136. Optionally, the rollers 126 may be rotated by one or more load motors 124 for this purpose. In this case, one or more sensors or switches 125 detects the presence of the biochip at detection system slot, causing the load motors 124 to turn on. The load motors 124 and rollers 126 provide a biochip mover for moving the biochip 22 horizontally. Alternate forms of biochip movers may be used instead of the load motors 124 and rollers 126, such as linear actuators, rack and pinion platforms, solenoids, etc. A biochip mover may be provided with single direction actuators and/or spring elements. The battery 28, the LEDs 46 and photodiodes 48 of the optical system, the jack screw motor 122 and the load motor 124 are electrically connected to a control board 130 of the electronic system 26, which controls the operations described below.

[0067] In use, cells 30 and water or medium are provided into the wells 72 of the microfluidic biochip 22. The seal layer 70, which may be a metal foil layer, is then applied over the fifth layer 60 to seal the wells 72. The microfluidic biochip 22 is then ready for use, although the microfluidic biochip 22 may optionally also be stored for days or weeks with the cells having sufficient water and nutrients to maintain life.

[0068] The detection system 20 is placed in the desired location. Since the detection system is compact and requires no external connections, the detection system may be used in wide variety of locations. The detection system 20 is turned on via the switch 132. The microfluidic biochip 22 is loaded into the detection system slot 136. The jack screw motor 122 is turned on, rotating the jack screws 120 which lowers the top plate 114 towards the microfluidic biochip 22. The tips of the water supply tube 96 and the vacuum tube 100 pierce through the foil layer 70 and engage into the water inlet 76 and the air outlet 78 of the microfluidic biochip 22, respectively. The vacuum pump 98 is turned on, drawing air through the air flow channel 84. The optical system 24 is also turned on. An extension tube may optionally be provided on the air inlet to better sample air from a specific location rather than sampling ambient air around the detection system.

[0069] VOC’s in the air drawn into the microfluidic biochip 22 pass through the membrane 65 and bind to an appropriate OR of the cells 30, transducing a signal that ultimately produces fluorescence when illuminated by the blue LED 46 or other light source reflected into the wells 72 by the mirror 44. When present, the fluorescence is detected by the photodiode 48. The detection event may then be displayed, transmitted and/or recorded.

[0070] With the tip of the water supply tube 96 engaged into the water inlet 76, water or other medium flows via capillary action from the water supply container 94 through the capillaries 80 and into the wells 72 to supply the cells 30. The cells 30 are consequently supplied with water from the capillaries 80, and are exposed to VOC’s passing through the membrane 65, but the cells 30 are otherwise sealed off from the environment.

[0071] When air sampling is completed, the microfluidic biochip 22 is removed or ejected from the detection system 20 and may be replaced with a new microfluidic biochip 22.

[0072] The detection system 20 may be provided with a biochip loader 150, together forming a combined unit 148, shown in Fig. 16, which can store multiple biochips 22 and automatically load and unload biochips 22 into and out of the detection system 20. The loader 150 allows the detection system 20 to operate unattended for an extended period of time. Figs. 13-15 show the loader 150 with no housing. Generally, the loader 150 is contained within a housing which may be similar to the housing 90 shown in Figs. 8-9. Alternatively, the loader 150 and the detection system 20 may be provided together in a single housing. In either case, the loader 150 is secured in a fixed position relative to the detection system 20, to allow biochips 22 to be moved between them. The loader 150 may also be electrically connected to the control board 130 or other component of the electronic system 26 of the detection system, with the control board 130 controlling both the detection system and the loader 150.

[0073] As shown in Figs. 13-14, the loader 150 has a frame 152 including guide posts 128 attached to a frame base 154 and a motor plate 158. A lift plate 166 is movable vertically on the guide posts, driven by jack screw motors 122 rotating jack screws 120. Bushings 168 allow the lift plate 166 to slide vertically on the guide posts 128 while reducing sliding friction and preventing lateral movement. A guideway 160 is formed within the frame 152 by columns 164 attached to the frame base 154 and the motor plate 158. The columns 164 pass through openings in the lift plate 166. The guideway 160 is configured to hold a stack of biochips 22 on the lift plate 166. A loader slot 180 is provided at the top of the guideway 160 to allow biochips 22 to be placed into the guideway 160. Fig. 13 shows a stack of 3 biochips 22 in the loader 150, although the loader may have capacity to hold e.g., 2-10 or more biochips 22. Fig. 13 which is a front view of the loader 150, shows the loader slot 180 formed by openings or cut away sections 182 through the upper ends of the front columns 164. The rear columns may have the same design, so that the loader slot 180 extends entirely through the guideway 160 from the front to the back of the loader 150.

[0074] A limit switch or sensor 174 may be located at the bottom of the guideway 160 to sense when the lift plate 166 is in the full down position. A camera 170 or other optical detector may be provided on the bottom side of the motor plate 158 to visually detect the presence and/or number of biochips 22 in the loader 150, and/or to read an identifier on a biochip, such as a bar code on the seal layer. Referring to Figs. 13-15, the loader 150 has a biochip mover, which may be provided in the forms of four load motors 124 on the motor plate 158. Each load motor rotates a roller 126, for moving biochips 22 into and out of the loader 150.

[0075] In use, the lift plate 166 of the loader 150 is lowered to or near the bottom of the guideway via the jack screw motors 122 rotating the jack screws 120. Multiple new or unused biochips 22 are inserted (by hand) through the loader slot 180 onto the lift plate 166 in the guideway 160. The biochips 22 may be keyed with the loader slot 180 so that the biochips can only be loaded in a single correct orientation. Alternatively the biochips 22 may have a projection or other feature that allows loading in only the single correct orientation. In the combined unit 148, the detection system 20 and the loader 150 are fixed in position (e.g., bolted into place in a housing or a mounting plate), with the front of the loader 150 facing the front of the detection system 20, and with the loader slot 180 of the loader adjacent to, and vertically and horizontally aligned with the detection system slot 136. In this design, the biochips 22 may be loaded into the loader 150 through the loader slot 180 at the back of the loader 150.

[0076] With the combined unit 148 placed or located in the desired room or space, the electrical system is turned on using the switch 132. The control board 130 confirms the presence of one or more biochips 22 in the loader 150, and optionally performs other functions, such as system checks, recording, reporting, etc. The control board activates the jack screw motors 122 to raise the lift plate 166 to vertically align the top-most biochip 22 with the loader slot 180. The load motors 124 of the loader 150 and the detection system 20 are turned on in the forward direction causing the rollers 126 to move the top-most biochip out of the loader 150 and into the detection system 20. The detection system 20 operates to detect VOC’s as described above.

[0077] The cells in an operating biochip 22 can effectively operate for several days, for example from 3 to 10 days. The duration of biochip operation is a function of the ability of the receptors (OR) to last, and not of cell viability. Cells with improved ORs may be able to operate longer than 10 days. The ORs in cells in a sealed biochip may be stored in the loader 150 for up to six weeks. Regardless of the OR effective duration, after a prescribed time interval, or after other factors determine the ORs are no longer operating sufficiently, the control board 130 initiates replacement of the used biochip 22. The load motors 124 are turned in the reverse direction, with the load motors of the detection system 20 causing the used biochip 22 to move out of the detection system 20 and back into the empty loader slot 180 in the loader 150. The load motors 124 of the detection system 20, also rotating in the reverse direction, move the used biochip 22 through the empty loader slot 180 and the used biochip is ejected out from the back of the loader 150 into a collection location. The control board operates the jack screw motors 122 to lift the lift plate 166 to vertically align the next biochip in the guideway 160 with the loader slot 180. The load motors 124 are again switched on in the forward direction moving the next biochip from the loader 150 into the detection system 20. This sequence is continued until all of the biochips 22 in the loader 150 have been used. Used biochips may be ejected out of the detection system and into a collection container as shown in Figs. 45 and 46. The control board may wirelessly communicate with a technician to provide detection results, and/or diagnostic and status data, or to allow the technician to remotely control operation of the combined unit 148.

[0078] The OR’s (olfactory receptors) may be sequences extracted from the human (600 ORs) and mouse (1300 ORs) genomes. Synthetic ORs with sequences that are not found in nature may be used. Such synthetic constructs are still considered ORs based on their sequence and functional similarities to natural ORs.

[0079] Cell types used include the Hana3A cell line, derived from the commonly used

HEK293 (human embryonic kidney) cells. This cell line contains accessory proteins that help the expression of ORs, such as receptor transporter proteins RTP1 , receptor expression enhancing proteins REEP1 and REEP2, as well as the protein Gaolf(s) necessary to transduce the signal. The second cell type which may be used is primary astrocytes, extracted from rat embryonic brains and expanded in vitro. Both cell types have been shown to function equally well in detecting VOCs.

[0080] The number of cells needed to generate a measurable response depends on the brightness of the cells and the sensitivity of the fluorescence detector. In the portable detection system 20 described, about 10,000 cells are used for each well. In the design shown in Fig. 12, the optical system has four optical paths, one for each well, with each optical path including the components as shown in Fig. 2.

[0081] In the example shown in Fig. 1 , band-pass filters 42 and a dichroic mirror 44 are used to separate excitation light from emitted light. The excitation source for each cell population may be a blue LED 46 (Nichia NSPB500AS) with a viewing angle of 15 degrees, coupled to a collimating lens (Thorlabs LB1157) and a blue excitation filter (Semrock FF01 469-35). The dichroic mirror (Semrock FF506) reflects the excitation light towards the cells in the biochip 22. A doublet of lenses focuses the excitation light onto the cells, and in turn collimates the emitted light back. Emitted light crosses the dichroic mirror and is filtered from scattered excitation light by a green emission filter (Semrock FF01 525-39). The filtered emitted light is focused by a lens on a silicon Photodiode 48 (Vishay VEMD5510C).

[0082] As shown in Fig. 2, the fluorescence reporter may be excited by blue light and emit green light. The conversion rate greatly increases (over 30 times) when the reporter is in the presence of calcium, which leads to an increase in emitted green light when the cells detect an odorant.

[0083] In order for the cells to generate a quick response, they are advantageously directly seeded on the membrane 65 that separates them from the outside environment. As it is difficult to embed electrodes on such thin membranes, the system monitors calcium flux in a contactless, optical way.

[0084] Fluorescence collected from one population depends on the number of cells and expression level of the calcium reporter protein. Cell number does not change as the system uses cells that do not divide. The number of functional fluorescent reporters in each cell can decrease over time due to natural protein turnover and photobleaching (light induced damage to fluorescent molecules). However the cells continuously produce new fluorescent proteins that compensate for this loss.

[0085] The fluorescence level is converted into a voltage by the photo-diode 48, and can easily be monitored or digitized for further processing. The change in fluorescence occurs at a timescale of a few seconds. At those low frequencies, the ambient electrical and optical noises affect the photo-diode voltage significantly more than the true fluorescence signal. This can be circumvented by providing the fluorescence signal a high frequency signature and filtering out the other frequencies. For example, the following steps may be used. 1 . flashing the excitation LED 46 at 6 kHz, which causes in turn the fluorescence emission to have the same frequency; 2. multiplying the raw fluorescence signal with a reference signal of same frequency and same phase. Since the product of two periodic signals tends to zero when their frequencies are different, most of the noise (which isn’t 6 kHz) is significantly attenuated; 3. smoothing the product with a low pass filter to remove the high frequency oscillations and only keep its DC component.

[0086] As shown in the example of Fig. 1 , the initial analog to digital converter (ADC) step is performed by a low noise electrophysiology chip (Intan RHD2132) originally designed to record action potentials. The digital lock-in amplifier is designed in Verilog and implemented on a SPARTAN6 FPGA board. The lock-in output can be displayed on a TFT screen connected to the FPGA board, or sent to an on-board computer (Raspberry Pi Zero) through a custom parallel communication protocol.

[0087] An on-board computer can perform live analysis in order to translate the raw fluorescence intensity into detection events. This processing may consist first in computing the mean and standard deviation of the derivative of the signal over the previous 30 seconds. Detection occurs if the instantaneous derivative is greater than the average derivative + C times the RMS ( dF > dF + C X (( dF - dF)²))^V2 for at least n seconds, with and being chosen to favor either accuracy or speed of detection.

[0088] The membrane 65 on which the cells are living provides the interface that separates the controlled cellular environment from the outside air. The membrane may advantageously allow VOCs to diffuse across the membrane in seconds; prevent bio contaminants from entering the cell medium and damaging the cells; be optically clear in order to visualize the cells; be chemically compatible with cell adhesion and growth; and be mechanically, chemically and heat resistant.

[0089] The membrane may be a thin (15 microns) PTFE (Teflon®) membrane with high porosity (75%) and a maximum pore size of 30 nm, which is smaller than bacteria

[0090] and most relevant viruses. The membrane may be opaque when dry, however after wetting of the membrane with a low surface tension fluid like IPA, the membrane becomes transparent and can be kept transparent as long as one side is kept in contact with IPA, water, or cell medium. In spite of its thinness, the membrane is sturdy and can be heated to more than 200 degrees Celsius, which allows coating it with an anti-stiction material on the external side for some applications while improving cell adhesion by treating it with plasma, and incubating it with poly-D-lysine on the inward-facing side. A silicon oxide (Si02) membrane may also be used. A pre-concentrator may be used to adsorb VOCs and desorb them upon heating.

[0091] Evaporation of water or medium through the membrane into the air flow channel 84 is inherently tied to air sampling. Medium evaporation is one of the main causes of failure in cell culture. As water evaporates, the concentration of dissolved substances, such as salts, increase up to the point that the cells cannot function properly. Counter-acting this phenomenon helps to keep the cells alive. Referring to Fig. 7, measured rates of evaporation in the biochip of Figs. 3-5 is on the order of 60 microliters per hour (40 mL/month for the biochip of Figs. 3-5). This value is significant in comparison to the volume of medium that is sufficient for the cells to survive for one month. Indeed, based on the rate at which cells consume nutrients, they only require a few hundred microliters of medium per month. Perfusing fresh medium can compensate for this evaporation, but is wasteful since cells need water rather than fresh medium. Flowever, perfusing pure water would flush away the vital solutes contained in the medium.

[0092] Thus the biochip is designed to use evaporation and capillary action inside the chip to aspirate water from a water supply container 94. If the water supply container is connected to the wells by thin enough capillaries 80, the speed of incoming water prevents solutes in the wells 72 from diffusing back into the water supply container, which insures that osmolarity remains constant inside the wells. The system also has the advantage to be self- regulated in a passive way. If the evaporation rate increases, the depression in the well will increase and draw water faster.

[0093] The vacuum pump 98 is driven by an electric motor which may use less than 0.5 W when on. There is no pump for water, as the transpirative osmolarity control system is passive. The vacuum pump 98 may run continuously, or intermittently, depending on the condition of the ORs and the status of the detection system.

[0094] As the example of Figs. 3-5 uses mammalian cells, the optimal temperature is

[0095] 37°C. Temperature control may be achieved by a single peltier module 140, attached to small aluminum overlay that distributes the heat over the four wells. The peltier element acts as a heat pump, transferring heat from one side of the unit to the other based on the direction of current flow through the device. An H-bridge circuit (DRV8838) may be used to control the current direction to either heat or cool the wells based on the temperature measured with an internal thermocouple (MAX31855). The temperature measurements and the control of the H-bridge are both performed by the on board computer.

[0096] Figs. 18-21 show an alternative biochip 222 designed to work with a saliva swab 250. A first end of the swab 250 is a handle 252 and a sponge section 254 is at the second end of the swab. An opening 256 is provided between the first end and the second end of the swab 250. The sponge section 254 may be attached to the handle 252 by two narrow spaced apart arms 258, so that the opening extends over 70 to 95% of the width of the swab 250.

[0097] As shown in Fig. 21 , the biochip 222 has a channel 234 in a bottom or base section 226. The channel 234 is dimensioned to receive the sponge section 254 of the swab 250 to introduce saliva (or other liquid) into the biochip 222. A membrane 232 covers or forms the top of the channel 234. A top section 224 is positioned on top of the membrane 232. The top section 224 has wells 230 containing cells. The cells are supported on and may grow on the top surface of the membrane 232. The upper ends of the wells 230 are sealed via a seal layer 228. A channel seal 236 seals off the channel opening. The seal layer 228 and the channel seal 236 may be a foil or plastic layer adhered to the top and bottom sections, respectively.

[0098] In use, the swab 250 is placed into the patient’s mouth, with the sponge section 254 absorbing saliva. As shown in Fig. 23, the sponge section 254 changes color after absorbing sufficient saliva. This provides a visual indication that the swab is ready for use in the biochip 222. As shown in Figs. 24 and 25, the swab 250 containing saliva is pushed through the channel seal 236 and into the biochip 222. The sponge section 254 slides along or otherwise comes into contact with the bottom surface of the membrane 232. Saliva passes from the sponge section 254 through the membrane 232 to the cells. The cells react as described above relative to Figs. 1-7 providing detection of a compound or metabolite.

[0099] As shown in Fig. 26, with the swab 250 fully inserted into the biochip 222, the sponge section 254 (which may be largely or entirely opaque) is moved to the back of the biochip, and the opening 256 is aligned under the wells 230. The optical detection system of Figs. 1-7 can therefore detect fluorescence from the cells in the wells. The height of the sponge section 254 may be greater than the height of the channel 234, and the membrane 232 may form the top surface of the channel 234 in the area under the wells. In this way, the sponge section 254 may resiliently push against and slide along the membrane 232 as the swab 250 is inserted into the biochip 222. This allows saliva to diffuse into and through the membrane and contact the cells in the wells. The swab 250 can of course also be used to introduce other liquids, besides saliva, into the biochip 220.

[0100] A locking mechanism 240 as shown in Fig. 27, if used, may lock the swab into the biochip 222. This allows for positive registration between each specific swab and a specific (e.g., coded or identified) biochip. The locking mechanism 240 may also be designed to provide a tactile indication that the swab 250 is properly positioned in the biochip 222 for optical detection.

[0101] Figs. 28-31 show another biochip 260 and swab 270. In this design, the swab 270 need not have any opening. This allows the swab 270 to be more compact than the swab 250. The swab 270 has a handle and an adjoining sponge section. As shown in Fig. 31 , in the biochip 260 the cells are contained in wells 230 between the membrane 232 on the bottom or base section 226 and a seal layer 228. A compressible layer 262 overlies the seal layer 228. Protrusions 264 project down from a protrusion plate 266. The protrusions 264 are aligned over the wells 230 of the biochip 260.

[0102] In use, the swab 270 collects saliva from the user’s mouth. The sponge section 254 changes color after absorbing sufficient saliva, as shown in Figs. 32 and 33. The swab 270 is inserted into a channel above the wells and below the compressible layer 262, as shown in Fig. 34. The detection system 20 detects the biochip. The motors move the top plate 114 down to engage the biochip 260, which at the same time causes the saliva sample on the sponge section 254 to move into the wells 230.

[0103] Specifically, the compressible layer 262 allows the time at which the saliva sample on the sponge is introduced to the cells in the wells 230 to correspond to the time at which the top plate 114 of the detection system 20 is moved downward to cause the tubing to make contact with the inlets of the biochip 260. The response of the biosensors to the saliva sample only lasts for a finite amount of time, so it is important that the introduction of the saliva sample to the cells occurs when the outputs can be read and analyzed. When the top plate 114 of the detection system 20 shown in Figs. 8 and 17 moves downward, it pushes the protrusions 264 on the protrusion plate 266 downward into the compressible layer 262, causing it to break the seal layer 228. This allows the sponge section of the swab 270 to make contact with the fluid inside of the wells 230. The compressible layer prevents the sponge from sliding outside of the desired bounds of the biochip, preventing the sponge from inadvertently contacting other surfaces in the detection system 20. Fluorescence from the cells may be detected from the bottom of the biochip 260.

[0104] Figs. 36-39 show yet another alternative biochip 280 and swab 290. In Fig. 39, the biochip 280 has a channel 284 in a top section 282. The wells 230 are also in the top section 282. A membrane 232 is positioned between the top section 282 and the bottom section 286. A channel seal 236 is placed over the channel 284 to prevent leakage out from the biochip 280. A top seal 228 seals the open top ends of the wells 230. [0105] In use, the sponge section 254 of the swab 290 changes color after absorbing sufficient liquid, as shown in Figs. 40 and 41 . Fig. 42 shows the swab 290 aligned with the channel 284 and ready to be inserted into the biochip 280. The swab 290 and the wells 230 are vertically aligned. Fig. 43 shows the swab 290 now piercing or breaking through the channel seal 236. The sponge section 254 is pushed into and/or past the wells 230. Saliva (or other liquid absorbed into the sponge section 254) moves into the wells and contacts the cells in the wells.

[0106] The height of the sponge section 254 may be greater than the height of the channel 284. In this case, as the sponge section 254 is inserted into the channel 284, it is compressed vertically. Then, as the sponge section 254 is pushed over the wells 230, it expands and contacts the fluid inside of the wells. The saliva sample on the sponge section 254 diffuses into the fluid in the wells and contacts the cells on the membrane. Or particulates on the sponge section can settle in the bottom of the wells and make contact with the cells. In some cases the sponge section may directly make contact with the cells.

[0107] The wells 230 in this embodiment may be formed as perforated tubes. As the sponge section 254 is pressed against the perforated tubes, liquid flows out of the sponge section 254, through the perforation holes in the sidewalls of the tubes, and into the wells 230. The sponge section 254 may have slots or thinned sections aligned with the wells, to allow the sponge section 254 to be more easily pushed through or past the wells. The biochip 280 may have a locking mechanism 240, as discussed above and as shown in Fig. 44.

[0108] The sponge section 254 may of course be made of various absorbent materials besides natural or synthetic sponge, such as fibrous and other materials, including transparent materials.

[0109] The bio chips shown in Figs. 21 , 31 and 39 may include design aspects of the biochip shown in Figs. 3-6, including materials used, assembly, dimensions, capillaries, inlet and alignment holes. These biochips may be manufactured in layers, as described above. The wells may be formed as round holes or bores through one or more layers. In general, the living cells are supported on the membrane, and the wells are partially or entirely filled with liquid. [0110] Thus systems and methods have been shown and described. Various changes and substitutions may of course be made without departing from the spirit and scope of the invention. The invention, therefore, should not be limited, except by the following claims and their equivalents.

Claims

1. A biochip, comprising: a first section having a channel; a membrane on top of the channel; a second section on top of the membrane; one or more wells in the second section for holding living cells; living cells on the membrane in the wells, the living cells fluorescing in the presence of a selected light and compound or metabolite; and at least a portion of the biochip transparent to allow light to enter the wells.

2. The biochip of claim 1 wherein the first section and/or the second section is transparent.

3. The biochip of claim 2 wherein a front end of the channel is sealed by a pierceable channel seal.

4. The biochip of claim 2 wherein the wells are formed by holes through the second section, further including a well seal on the second section over the holes.

5. The biochip of claim 1 wherein the membrane comprises PTFE.

6. The biochip of claim 1 further including a gas inlet and a gas outlet extending vertically from a top surface of the biochip to a first end and a second end, respectively, of the channel.

7. A swab for use with a biochip, comprising: a handle section attached to an absorbent section by first and second arms; and an opening through the swab between the first and second arms.

8. The swab of claim 7 wherein the first and second arms are on opposite sides of the opening.

9. The swab of claim 8 wherein the opening has a width equal to at least 70% of the width of the absorbent section.

10. The swab of claim 8 wherein the absorbent section comprises a sponge material having a rectangular cross section.

11. A kit for testing a liquid, comprising: a biochip and a swab adapted to absorb a liquid and for insertion into the biochip; the biochip containing living cells; at least a portion of the biochip transparent to provide a light path from the living cells out of the biochip; and the swab having an opening in the light path when the swab is inserted into the biochip.

12. The kit of claim 11 wherein the living cells fluoresce in the presence of a selected light and compound or metabolite.

13. The kit of claim 11 with the biochip having a channel and the swab having an absorbent section adapted for insertion into the channel.

14. The kit of claim 13 with the biochip having a membrane, a first side of the membrane forming a top surface of the channel, and the living cells on a second side of the membrane.

15. The kit of claim 11 further including a locking mechanism for locking the swab into engagement with the biochip.

16. A biochip comprising: one or more wells for holding living cells; a membrane closing off a first end of each of the wells; a channel over the membrane for receiving an absorbent section of a swab; protrusions on a protrusion plate over the channel, each protrusion aligned with one of the wells, the protrusions movable to drive a liquid sample in the absorbent section of the swab into the wells.

17. The biochip of claim 1 wherein at least a portion of the biochip is transparent to allow light to enter the wells.

18. The biochip of claim 16 further including a compressible layer between the protrusions and the membrane.

19. The biochip of claim 16 wherein the wells are formed in a first section of the biochip made of a transparent material.

20. The biochip of claim 19 further including a seal layer closing off a second end of each of the wells, wherein movement of the protrusion plate pierces the seal layer.

21. The biochip of claim 20 wherein a first side of the membrane forms a top surface of the channel, and further including living cells on a second side of the membrane.

22. The biochip of claim 21 wherein the living cells fluoresce in the presence of a selected light and a selected compound or metabolite.

23. A biochip, comprising: a first section; a second section joined to the first section; a membrane between the first section and the second section; one or more wells in the second section for holding living cells; and a channel in the second section intersecting the wells.

24. The biochip of claim 23 wherein at least one of the first section and the second section is transparent.

25. The swab of claim 7 wherein the absorbent section changes color after absorbing a selected amount of liquid.

26. A testing method, comprising: collecting a saliva sample from a subject’s mouth by inserting an absorbent section of a swab into the mouth; inserting the absorbent section of the swab into a biochip containing living cells; detecting a reaction of the living cells to the saliva; and identifying a compound or metabolite in the saliva based on the reaction of the living cells.

27. The testing method of claim 26 wherein the reaction is fluorescence.

28. The testing method of claim 26 further including sliding the absorbent section over a first side of a membrane in the biochip, wherein the living cells are on a second side of the membrane.

29. The method of claim 26 wherein the absorbent section of the swab is inserted into a channel of the biochip, the absorbent section having a height greater than the height of the channel, so that the absorbent section of the swab is compressed as it is inserted into the channel, and wherein a portion of the absorbent section expands at least partially into one or wells in the biochip, to bring the saliva sample into contact with the cells.