Sensing tip with electrical impedance sensor

Abstract

A sensing tip including a pipette tip having a cavity which communicates with an external environment of the pipette tip through an aperture located at a distal end of the pipette tip, and an impedance sensor having a sensing area including at least two electrodes located respectively outside and inside the pipette tip, wherein the sensing area is arranged within the aperture.

Claims

1. A sensing tip having a longitudinal axis, the sensing tip comprises: a pipette tip; a membrane coated in the pipette tip, wherein the membrane has side walls and has a flat dispensing face at a distal end of the pipette tip, wherein the flat dispensing face of the membrane is substantially perpendicular to the longitudinal axis of the sensing tip; a cavity inside the pipette tip communicates with an external environment of the pipette tip through an aperture that is located in the flat dispensing face of the membrane; and an impedance sensor having a sensing area and having at least two electrodes located respectively outside and inside the pipette tip, wherein the electrode located inside the pipette tip is not in contact with the side walls of the membrane, wherein the electrode located outside the pipette tip is not in contact with the flat dispensing face of the membrane, wherein the sensing area is arranged within the aperture.

2. The sensing tip according to claim 1, wherein the impedance sensor is adapted to be used as a Coulter counter.

3. The sensing tip according to claim 1, wherein the membrane is made of a different material than a material of the pipette tip.

4. The sensing tip according to claim 3, wherein the flat dispensing face of the membrane includes a dielectric membrane.

5. The sensing tip according claim 1, wherein the aperture is a slotted hole.

6. A method of using a sensing tip, the sensing tip having a longitudinal axis, and the sensing tip comprises a pipette tip, membrane coated on the pipette tip, wherein the membrane has side walls and a flat dispensing face at a distal end of the pipette tip, wherein the flat dispensing face is substantially perpendicular to the longitudinal axis of the sensing tip, a cavity communicating with an external environment of the pipette tip through an aperture located in the flat dispensing face of the membrane, and an impedance sensor having a sensing area including at least two electrodes located respectively outside and inside the pipette tip, the electrode inside the pipette tip not being in contact with the side walls of the membrane, and the electrode outside the pipette tip not being in contact with the flat dispensing face of the membrane, and wherein the sensing area is within the aperture, the method comprising the step of: detecting particles within a liquid flowing through the aperture within the sensing area at a single-particle resolution to determine at least one of number and size of the particles.

7. The method according to claim 6, further comprising the step of: assessing at least one of cell viability, membrane properties, and cell properties of the particles.

8. The method according to claim 6, further comprising the step of: analyzing a particle density on the particles or a subset of particles.

9. The method according to claim 6, further comprising the step of: detecting a control set of cells within a solution of particles.

10. The method according to claim 6, further comprising the step of: sequentially dispensing a subset of cells previously taken up in a solution of particles.

11. The method according to claim 6, wherein a set of cells or subset of cells is composed of a single particle.

12. The method according to claim 6, further comprising the step of: sequentially dispensing a set or a subset of particles with a resolution of single particle.

13. The sensing tip according to claim 3, wherein the external electrode is located on a side wall of the membrane.

14. The method according to claim 6, wherein the external electrode is located on a side wall of the membrane.

15. The sensing tip according to claim 1, wherein the electrode inside the pipette tip is floating.

16. The method according to claim 6, wherein the electrode inside the pipette tip is floating.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Cross sectional view of the extremity of a sensing tip according to the invention (wire inner electrode mode)

(2) FIG. 2: Cross sectional view of the extremity of another tip according to the invention (thin film inner electrode mode)

(3) FIG. 3: Pipette operating with a sensing tip according to the invention

(4) FIG. 4: A first exploded view of a sensing tip according to the invention

(5) FIG. 5: A second exploded view of the tip of FIG. 4

(6) FIG. 6: A specific shape of the aperture allowing for the automatic unclogging

REFERENCE NUMERALS USED IN THE FIGURES

(7) 1 conventional tip

(8) 2 dielectric membrane

(9) 3 aperture

(10) 4 external electrode

(11) 5 internal electrode

(12) 6 wire internal electrode (optional mode)

(13) 7 instrument

(14) 8 sensing tip

(15) 9 solution

(16) 10 screen

(17) 11 circular hole

(18) 12 slotted hole

(19) Tip Structure

(20) The sensing tip illustrated in the examples of FIGS. 1 and 2 includes a conventional tip 1 covered with a dielectric membrane 2 at the tip distal end. The membrane 2 is provided with an aperture 3. An outer electrode 4 is placed on the membrane and an inner electrode 5 (FIG. 2) or 6 (FIG. 1) is located within the conventional tip 1.

(21) Exploded views of those sensing tips are illustrated on FIGS. 4 and 5.

(22) The electrodes 4 and 5 are used to establish a determined electric field. In a conductive medium, for instance a solution containing particles (e.g. cells), a current flows between the inner (5,6) and the outer (4) electrodes. Because of this design both current and particles are forced to flow through the aperture 3. As a consequence, the current is mostly influenced by the particle in the aperture 3 according to coulter counter principle. Knowing the electrical field and measuring the current, impedance spectroscopy or coulter counting can be performed on the particles in the aperture 3. For this purpose a time-resolved impedance analyzer is connected to the electrodes 4 and 5 or alternatively 4 and 6. Hence dielectric and structural properties of the particle in the aperture 3 can be measured.

(23) The design of the aperture 3 must be fine-tuned with respect to the particle to be analyzed.

(24) In particular, it is best to adapt the diameter of the aperture 3 to the particle diameter to avoid clogging while maximizing signal-to-noise ratio.

(25) It is best to adapt the membrane 2 thickness to maximize time-of-travel of the particle in the aperture 3 while maximizing the signal-to-noise ratio.

(26) Electrodes 4, 5 or 6 should have a sufficient area to maintain the current at the frequency of interest for the measure.

(27) Electrode 4, 5 or 6 can be attached to the structure or can be left floating. However, it is best to work with electrode 4, 5 or 6 as close as possible to the aperture 3 as it allows to work with a minimal wetting of the tip.

(28) Tip Fabrication

(29) Using a membrane 2 allows to better control the aperture 3. The membrane 2 is preferably coated on the conventional tip 1 distal end using standard chemical vapor deposition (CVD) process. The coating is a pinhole-free and conformal polymer (such as poly(p-xylylene with tradename Parylene). It is deposited with accurate thickness ranging from 50 nm to 1 mm depending on the application. A biocompatible polymer is used to handle biological sample (Parylene USP class VI). Prior to chemical vapor deposition, tip 1 is filled with a sacrificial plug. The plug is made of a soluble polymer, for instance poly(ethylene-glycol) which is soluble in water. After CVD, the polymer closes the opening of the tip. The plug is dissolved in solvent. Thus, the polymer forms a membrane that closes tip 1. Alternatively, membrane 2 is made of a polymer material which is bonded to tip 1, for instance by ultrasonic welding or other chemical bonding. Alternatively, tip 1, membrane 2 and aperture 3 are molded as a single element.

(30) An aperture 3 is opened through membrane 2 at the extremity of tip 1. This process is performed by laser ablation, punching, etching or drilling. The diameter of aperture 3 ranges from 50 nm to 1 mm.

(31) The aperture 3 may be a simple circular hole 11 or may be formed by more complex shapes such as a slotted hole 12 to prevent the aperture 3 from clogging (see FIG. 6).

(32) Electrodes are deposited inside and outside the conventional tip 1 in order to enable the creation of an electric field trough aperture 3. The inner and outer electrodes are made of metal or conductive material deposited on the surface of tip 1 by sputtering or any other deposition method. The inner electrode can also be a conductive wire 6.

(33) Instrument

(34) The sensing tip 8 may be coupled with an instrument (see FIG. 3) that is arranged to connect to the proximal end of the tip 8. The instrument is composed at least of a fluidic apparatus, an electrical impedance analyzer and a control unit. The fluidic apparatus controls the flow of the liquid inside the tip, which may be stopped, flowed inside or flowed outside the tip. The electrical impedance analyzer measures the impedance provided by the sensor of the sensing tip. The control unit communicates with the electrical impedance analyzer and the fluidic apparatus. The control unit interprets the data sent by the electrical impedance analyzer and in response controls the fluidic apparatus. The control unit may have a user-interface to receive orders from the user and it may transfer the data on a display screen and/or to a computer for further analysis. The instrument can be as example an adapted pipette, multi-pipette or pipetting robot as well as any other instrument including the functions mentioned above.

(35) Examples of Use

(36) In a preferred embodiment, the sensing tip 8 is used to take up a controlled set of particles within in a solution 9. When passing the sensor, particles are counted and analyzed in term of number and size. Dielectric and structural properties of biological samples are analyzed to assess cell viability, membrane properties, granularity and any other relevant biological information. The sensing tip is operated with an instrument 7, such as a pipette as example, including a fluidic system, an embedded electronic and a display 10. The sensing tip is connected to the instrument 7. The tip extremity is immerged in solution 9 containing particles. The user presses a button on the display of the instrument to start taking up the particles in the solution. The fluidic system of the instrument generates a negative pressure that aspirates the particles. Particles flowing in the tip are detected and analyzed by impedance measurement when they pass through the sensor placed at the tip end. Impedance measurements are processed on the instrument and displayed on screen 10. The data can also be transferred on other computers or portable devices.

(37) In another embodiment, the sensing tip is used to dispense a part or all particles previously taken up in a solution according to preferred embodiment. Particle analysis is performed the same way as described in the preferred embodiment. Particle dispensing is performed by first immerging the tip either in initial container 9 or any container with fresh solution. The user presses a button on the display of the instrument to start dispensing the particles. The fluidic system of the instrument generates a positive pressure that flows the particles out. Particles flowing out the tip are detected and analyzed by impedance measurement when they pass through the sensor placed at the tip extremity. Impedance measurements are processed on the instrument 7 and displayed on a screen 10. The data can also be transferred on other computers or portable devices

(38) In another embodiment, the sensing tip is used to sequentially dispense a subset or all particles previously taken up in solution 9 according to preferred embodiment. The number of particles to be dispensed is defined by the user. It can dispense as few as a single particle at a time. The particles can be transferred from one container to another container or to other containers with no particle loss during the transfer. Particle dispensing is performed by first immerging the tip either in initial container 9 or any container with fresh solution. This step can be repeated for each new dispensing of particles. The user defines the number of particles to be dispensed at each sequential dispensing. The user presses a button to start dispensing the particles. The fluidic system of the instrument generates a positive pressure that flows the particles out. Particles flowing out the tip are detected and analyzed by impedance measurement when they pass through the sensor. A feedback loop on flow rate stops the dispensing once it reaches the number of particles to be dispensed. Impedance measurements are processed on the instrument and displayed on a screen. The data can also be transferred on other computers or portable devices.