WO2018210266A1

WO2018210266A1 - Flexure-guided piezo drill with large axial vibration and small lateral vibration

Info

Publication number: WO2018210266A1
Application number: PCT/CN2018/087080
Authority: WO
Inventors: Yu Sun; Wesley Johnson; Jun Liu; Changsheng DAI
Original assignee: Jiangsu Jitri Micro-Nano Automation Institute Co., Ltd
Priority date: 2017-05-18
Filing date: 2018-05-16
Publication date: 2018-11-22
Also published as: CN110719952B; CN110719952A; CA3091527A1

Abstract

A flexure (2)-guided piezo drill is described to generate large axial vibration and small lateral vibration. The flexure (2) mechanism is used to guide the trajectory of the micropipette (1) in the axial direction, and the resonant frequencies that result in large lateral vibration are removed from the driving pulses. The flexure (2)-guided piezo drill is particularly suited for penetrating the zona pellucida (ZP) of oocytes and embryos effectively and with small deformation.

Description

A flexure-guided Piezo Drill with Large Axial Vibration and Small Lateral Vibration

Field of the Invention

The present invention relates to an inertial impact drill, and more specifically, relates to applications in oocyte and embryo manipulation for in vitro fertilization.

Background of the Invention

Penetrating the zona pellucida (ZP) is an important step of in vitro fertilization (IVF) , with applications such as intracytoplasmic sperm injection (ICSI) and biopsy for preimplantation genetic screening (PGS) . The ZP is a thick, highly elastic layer composed of glycoproteins surrounding an oocyte or embryo.

In oocyte and embryo manipulation in IVF clinics, severely deforming the oocyte or embryo (e.g., 50 μm) is required for ZP penetration even when a sharp micropipette is used. Large oocyte deformation can damage oocyte spindles and lead to failure of oocyte fertilization and embryo development. Large embryo deformation can also affect subsequent embryo development. The use of piezo-actuated vibrations can improve ZP penetration without causing large oocyte or embryo deformation. Piezo drills are inertial impact devices using piezoelectric actuators to generate high frequency axial vibrations of a micropipette to induce local rupture of the ZP.

Current piezo drill designs cause cell damage by undesired large lateral vibrations on the micropipette tip. To help dampen lateral vibrations, a segment of mercury is often used close to the tip of the micropipette. The use of mercury in direct contact with biomaterials causes concerns in clinical practice as well as in biology research, which is a major hurdle of existing piezo drill devices for entering IVF clinics.

Commercial piezo drills from companies such as Prime Tech and Burleigh have the design in which the piezo actuator is located far from the micropipette and produces large lateral vibrations on the micropipette tip. Placing the piezo actuator directly behind the micropipette holder can help better focus the vibration at the micropipette tip. It was reported that this design configuration reduced the lateral vibration to around 20 μm (H.B. Huang, H. Su, H. Chen, and J.K. Mills, “Piezoelectric driven non-toxic injector for automated cell manipulation, ” Medicine Meets Virtual Reality Conference, pp. 231–235, 2011) as opposed to over 100 μm in the commercial design. However, the latter design still induces rather large lateral vibration of 20 μm and causes oocyte deformation to be larger than 10 μm.

Prior patents having relevance to our invention include US6251658B1 by Burleigh Instruments, US20080213899A1 by the University of Connecticut, US20110193510A1 by Newcastle Innovation, US5225750A and US5229679A by Prima Meat Packers, US20090069712A1 by Piezo Resonance Innovations, US5877008A by Lockheed Martin Energy Systems, and US20030059936A1 by Micronas GmbH. The following three are considered most relevant to our invention.

“Inertial impact drill for cytological applications” , US6251658B1 by Burleigh Instruments, disclosed an inertial impact drill using opposing piezoelectric or electrostrictive actuators to drive the movement of an inertial mass and to produce the vibration of the micropipette or the microelectrode. No flexure mechanism as used in our invention was disclosed. Our invention also does not use two opposing piezoelectric actuators to produce vibrations.

“Rotationally Oscillating Injector” , US20080213899A1, disclosed an injector that penetrates an oocyte by rotating the micropipette with a rotary motor. Our invention does not involve rotation or the use of a rotary motor.

“Positioning system and method” , US20110193510A1, disclosed a nanopositioning stage using piezoelectric actuators and flexure mechanisms for accurate positioning. Although our invention also uses a piezoelectric actuator and a flexure mechanism, our device was designed to produce vibratory motions for penetrating the zona pellucida rather than accurate smooth positioning.

Summary of Invention

The device disclosed in this application is a piezo drill capable of generating large axial vibration and small lateral vibration. The piezo drill can be used to penetrate the zona pellucida (ZP) of oocytes and embryos effectively with small deformation.

For the above purpose, the invention utilizes the following technical solutions:

In one aspect, the invention provides a flexure-guided piezo drill apparatus for generating large axial vibration and small lateral vibration for zona pellucida (ZP) penetration with small oocyte or embryo deformation.

Preferably, said piezo drill has a micropipette, a flexure, a piezoelectric actuator, a flexure holder and a holding rod.

More preferably, said flexure comprises a central part, multiple flexure beams, an outer part and a flexure base.

Preferably, said flexure base is connected to the central part.

Preferably, said flexure is manufactured with stainless steel by wire electrical discharge machining.

Preferably, said micropipette is fixed on the flexure and can be easily replaced.

In a preferable embodiment, said central part of the flexure is connected to a tubing to provide negative pressure for oocyte or embryo aspiration, and the tubing is further connected to a pneumatic or hydromantic pump.

Preferably, the piezoelectric actuator is integrated on said flexure base.

Preferably, said piezoelectric actuator is fastened against the flexure base and preloaded with a screw and a metal shim.

Preferably, said outer part of the flexure is clamped by the flexure holder and fastened by two screws.

Preferably, said flexure holder is connected to the holding rod.

Preferably, said holding rod is mounted onto a micromanipulator, said micromanipulator having motion stages to achieve accurate positioning.

Preferably, said flexure beams are connected to the central part and the outer part of the flexure by double hinges.

Preferably, said hinges are elliptical hinges.

The present invention also discloses a mechanical design method that uses a flexure mechanism to guide the micropipette motion along the axial direction.

In another aspect, the design of driving pulses is described. The designed driving signals are pulses with resonant frequencies of the micropipette and the flexure attenuated.

Preferably, driving pulses for driving the piezo drill is processed by filtering out resonant frequencies of the micropipette and the flexure from the frequency spectrum of the driving pulses.

Brief Description of the Drawings

A detailed description of one or more embodiments is provided herein below by way of example only and with reference to the following drawings, in which:

FIG. 1 illustrates a schematic of the piezo drill.

FIG. 2 illustrates a top down view of the piezo drill showing tubing assembly.

FIG. 3 illustrates a schematic of the micropipette in contact with an oocyte or embryo and its equivalent mechanical system.

FIG. 4 illustrates a schematic of the flexure beam configuration and its double elliptical hinges.

FIG. 5 illustrates the filtered driving pulses for the piezoelectric actuator.

FIG. 6 illustrates the vibration of the micropipette mounted on the piezo drill measured by SEM.

FIG. 7 illustrates the axial vibration amplitude of the micropipette measured by the vibrometer.

FIG. 8 illustrates ZP penetration with small oocyte deformation using the piezo drill.

Detailed Description of the Invention

The present invention includes a mechanical design method for a piezo drill to generate large axial vibration and small lateral vibration, and a driving pulses design approach for driving the piezoelectric actuator.

A. Mechanical Design

As shown in FIG. 1 and 2, the flexure 2 comprises a central part 3, flexure beams 4, outer part 5, and flexure base 6. The flexure-guided design has a piezoelectric actuator 7 integrated on the flexure base 6. The motion of the micropipette 1 is guided by a number of compliant flexure beams 4. The central part 3 and outer part 5 are connected by the flexure beams 4 through elliptical hinges. The flexure base 6 is connected to the central part 5. The outer part 5 is clamped by the flexure holder 8. The flexure holder 8 is connected to a holding rod 9, and the holding rod 9 is mounted on a micromanipulator (not shown) .

The flexure 2 can be manufactured from a single piece of stainless steel by wire electrical discharge machining (EDM) . Thus, its monolithic structure does not suffer from nonlinearities caused by friction, wear and backlash.

Control over the actuation direction depends on the stiffness ratios between the lateral axes (Y and Z) and the axial axis (X) . By achieving a low X-axis stiffness with respect to the other two axes, the motion of the micropipette can be guided in the axial direction irrespective of slight misalignment during device assembly.

A dynamic model of the micropipette is first established. The objective is to ensure that the micropipette's lateral resonant frequencies are avoided in the design of flexures and the operational pulse train. FIG. 3 (a) shows a schematic of the micropipette in contact with an oocyte or embryo. FIG. 3 (b) shows the equivalent mechanical system. The micropipette tip’s equation of motion is derived using Euler-Bernoulli beam theory, and the micropipette’s resonant frequencies are obtained by analytically solving the equation.

The piezo drill uses a flexure guidance mechanism. The flexure mechanism uses elastic deformation of the material to deliver motion. For flexure design, the inner section of a flexure beam 4 is thickened to strengthen its out-of-plane (Z) stiffness, which results in the double hinged beam configuration shown in FIG. 4. In comparison, circular hinges have a high out-of-plane stiffness but a low motion range. Corner filleted hinges have a large motion range but a poor out-of-plane stiffness. Elliptical hinges combine these properties allowing for a stiff flexure while maintaining a relatively low stress at maximum extension for the flexure to have a long fatigue life and are chosen in our design. For the chosen flexure beam configuration, the six parameters of the flexure beams that need to be determined are the number of beams, beam thickness B _T, beam length B _L, hinge shape, hinge pivot point thickness H _T, and hinge length H _L.

Stainless steel was chosen to construct the piezo drill device because of its high yield strength, which is significantly higher than the predicted maximum stress encountered during vibration. Apiezoelectric actuator 7 is integrated on the flexure base 6 and is preloaded with a screw and a metal shim. Sufficiently preloading the piezo is important for ensuring its stability and avoiding brittle fracture at operating frequencies approaching or above the flexure's resonant frequencies.

The X-axis resonant frequency must be higher than the input pulse's frequency to avoid initiating flexure or micropipette resonance. However, too high an X-axis resonant frequency requires a high X-axis stiffness. Low stiffness ratios of the X-axis to the other axes can cause large off-axis motion and large lateral vibration. With the selected X-axis resonant frequency and the preload, iterative finite element structural simulation is conducted to determined flexure parameters.

B. Driving Pulses Design

Piezo drills use a pulse train to penetrate the ZP of an oocyte or embryo with reduced energy transfer for less oocyte damage. A driving pulse train is formed by applying multiple pulses per second with intervals among them; thus, it contains less energy compared to continuous driving signals.

Assuming the base frequency of the driving pulses is 18 kHz, and the resonant frequencies of the micropipette and the flexure include 3-7 kHz and above 20 kHz. A continuous sinusoidal signal with a frequency of 18 kHz has all of its spectral power at one point [see FIG. 5 (a) (b) ] . FIG. 5 (c) shows a pulse with a base frequency of 18 kHz. Its frequency response [FIG. 5 (d) ] contains values from all frequencies including 3-7 kHz and above 20 kHz which can cause lateral resonance.

A band-stop filter with cutoff frequencies of [3 kHz, 7 kHz] and a low-pass filter with a cutoff frequency of 20 kHz were used to filter the original pulse signal shown in FIG. 5 (c) . Both filters are second order infinite impulse response (IIR) filters.

The filtered pulse signal is shown in FIG. 5 (e) (f) . It can be seen that this filtered pulse has the undesired frequency ranges of 3-7 kHz and above 20 kHz significantly attenuated.

When the piezo drill device is used in oocyte/embryo ZP penetration, the filtered pulse shown in FIG. 5 (e) is applied multiple times within a second (e.g., 100 pulses) , which forms a pulse train, to the piezo actuator as the driving signal for ZP penetration. The amplitude of the driving pulse can be altered by varying the peak voltage.

Example 1

Materials:

A scanning electron microscopy (SEM, SU3500, Hitachi) was used to characterize the micropipette’s axial and lateral vibration amplitudes. A laser Doppler vibrometer (OFV-5000, Polytec) was used to verify the micropipette's axial vibration amplitude measured by SEM. The displacement resolution of the vibrometer can reach 1 pm.

Results:

SEM imaging has a low bandwidth (20 Hz) ; thus, as the micropipette vibrates, its vibrational envelope appears in SEM imaging as blurred edges [see FIG. 6 (b) (c) ] . Measuring the distance between an edge of the micropipette and its corresponding vibration-caused blurry edge enabled the quantification of the micropipette's axial and lateral vibration amplitudes.

FIG. 6 (b) (c) correspond to 15 kHz and 18 kHz, respectively, which are the frequencies of the driving pulse supplied to the piezoelectric actuator. The peak voltage of the driving pulse was held constant at 20 V. When 15 kHz driving pulses [FIG. 6 (b) ] were applied to the piezo actuator, the micropipette tip had a lateral vibration amplitude of 500 nm and an axial vibration amplitude of 1.2 μm. When the driving pulse's frequency was increased to 18 kHz [FIG. 6 (c) ] , the micropipette's axial vibration amplitude was still approximately 1.2μm; however, the lateral vibration amplitude increased to 2 μm (vs. 0.5 μm produced by 15 kHz driving pulses) . With 18 kHz driving pulses, the axial vibration amplitude measured by the laser doppler vibrometer was 1.2 μm [see FIG. 7] , in agreement with the SEM measured results. In comparison, existing piezo drills all have a large lateral vibration amplitude (>20 μm) and a very low axial vibration amplitude (<0.1 μm) .

Example 2

Materials:

Mouse oocytes were gathered from the Canadian Mouse Mutant Repository in the Toronto Centre for Phenogenomics. An inverted microscope (Nikon Ti, Nikon Microscopes) and a CCD camera (acA 1300-30gm, Basler) were used to observe the oocytes.

Results:

The success rate of ZP penetration with the piezo drill was 100%based on the testing of 45 mouse oocytes. FIG. 8 (a) (b) , corresponding to supplied driving pulses of 18 kHz and peak voltage of 20 V, show that the piezo drill is capable of penetrating the ZP of mouse oocytes with an oocyte deformation as small as 3.4 μm. In comparison, existing piezo drills produce mouse oocyte deformations larger than 10 μm unless a drop of mercury is used in the micropipette for damping.

Claims

A flexure-guided piezo drill apparatus for generating large axial vibration and small lateral vibration for zona pellucida (ZP) penetration with small oocyte or embryo deformation.
The apparatus of claim 1, wherein said piezo drill has a micropipette, a flexure, a piezoelectric actuator, a flexure holder and a holding rod.
The apparatus of claim 2, wherein said flexure comprises a central part, multiple flexure beams, an outer part and a flexure base.
The apparatus of claim 3, wherein said flexure base is connected to the central part.
The apparatus of claim 2, wherein said flexure is manufactured with stainless steel by wire electrical discharge machining.
The apparatus of claim 2, wherein said micropipette is fixed on the flexure and can be easily replaced.
The apparatus of claim 3, wherein said central part of the flexure is connected to a tubing to provide negative pressure for oocyte or embryo aspiration, and the tubing is further connected to a pneumatic or hydromantic pump.
The apparatus of claim 3, wherein the piezoelectric actuator is integrated on said flexure base.
The apparatus of claim 8, wherein said piezoelectric actuator is fastened against the flexure base and preloaded with a screw and a metal shim.
The apparatus of claim 3, wherein said outer part of the flexure is clamped by the flexure holder and fastened by two screws.
The apparatus of claim 2, wherein said flexure holder is connected to the holding rod.
The apparatus of claim 2, wherein said holding rod is mounted onto a micromanipulator, said micromanipulator having motion stages to achieve accurate positioning.
The apparatus of claim 3, wherein said flexure beams are connected to the central part and the outer part of the flexure by double hinges.
The apparatus of claim 13, wherein said hinges are elliptical hinges.
The apparatus of claim 1, wherein driving pulses for driving the piezo drill is processed by filtering out resonant frequencies of the micropipette and the flexure from the frequency spectrum of the driving pulses.