US-12618765-B2 - Cytometer and a method of cytometry

US12618765B2US 12618765 B2US12618765 B2US 12618765B2US-12618765-B2

Abstract

A cytometer includes a plurality of measurement sections, each comprising first and second sidewalls and a base therebetween, the sidewalls and base defining a channel portion extending from an entrance to an exit; an electrode group arranged on the opposite sides of the base and part way between the entrance and exit, the electrode group comprising an upstream electrode, a centre electrode and a downstream electrode in the way of a trajectory between the entrance and exit, and in respective order; the measurement sections being connected together to form at least one measurement channel comprising a plurality of the channel portions connected together in series; and, a lock-in amplifier; the central electrode of each group connected to the excitation signal port of the lock-in amplifier, and the upstream and downstream electrodes connected to the voltage differential input ports of the lock-in amplifier.

Inventors

Hiu-wai Raymond LAM
Wei Huang

Assignees

CITY UNIVERSITY OF HONG KONG

Dates

Publication Date: 20260505
Application Date: 20230127

Claims (14)

1 . A cytometer comprising a plurality of measurement sections, each measurement section comprising first and second sidewalls and a base extending therebetween, the first and second sidewalls and base defining a channel portion therebetween, the sidewalls and base extending from an entrance to an exit; an electrode group arranged on the opposite side of the base to the channel portion and part way between the entrance and exit, the electrode group comprising an upstream electrode, a downstream electrode and a center electrode, the electrodes being arranged such that as one travels from the entrance to the exit one passes over the upstream electrode, then the center electrode and then the downstream electrode; the channel portion having a width measured in a direction normal to the sidewalls; the measurement sections being connected together to form at least one measurement channel comprising a plurality of the channel portions connected together in series, with each channel portion of the measurement channel having a different width; and, at least one lock-in amplifier; the electrode groups being configured such that for each group the central electrode is connected to the excitation signal port of a lock-in amplifier and the upstream and downstream electrodes are connected to the voltage differential input ports of the lock-in amplifier.
2 . A cytometer as claimed in claim 1 wherein the at least one measurement channel comprises first and second channel portions only.
3 . A cytometer as claimed in claim 1 , wherein the at least one measurement channel comprises at least three channel portions.
4 . A cytometer as claimed in claim 1 , wherein the measurement sections are connected together to form a plurality of measurement channels, each measurement channel comprising a plurality of channel portions.
5 . A cytometer as claimed in claim 4 , wherein for at least two measurement channels the electrode groups of one measurement channel are connected to the corresponding electrode groups of the other measurement channel.
6 . A cytometer as claimed in claim 1 , further comprising a signal processor configured to receive the output from the at least one lock-in amplifier and perform the steps of (a) identify a set of ripples in the output which are produced by passage of a cell along different channel portions of a single measurement channel; (b) for at least two ripples α, β of the set of ripples which correspond to passage of the cell along channel portions cα and cβ determine the peak-to-peak amplitudes V pp α and V pp β of the ripples; and, (c) solve the simultaneous equations V pp α =k ( D deform 2 ( D cell ,W cα )+ b ) V pp β =k ( D deform 2 ( D cell ,W cβ )+ b ) to determine a cell surface charge k and the undeformed diameter of the cell where b is a constant, W cα and W eβ are the widths of the channel portions α and β and D deform (D cell ,W) is the diameter of a deformed cell of undeformed diameter in a channel of width and is related to D cell and W by the relation 4 3 ⁢ π ⁡ ( D cell 2 ) 3 = 2 ⁢ π ⁡ ( D deform 2 ) 2 ⁢ ( W 2 ) - 2 ⁢ π 3 ⁢ ( W 2 ) 3
7 . A cytometer as claimed in claim 6 , wherein the signal processor is further configured to perform the steps of (d) selecting a ripple r from the set of ripples and for the ripple r divide a predetermined constant C by the peak-to-peak time t pp of the ripple r to determine the speed of the cell which caused the ripple; (e) determining driving force F d on the cell in channel portion from a model for channel portion C r of the driving force as a function of cell speed and D cell ; and (f) determine cell stiffness E from D cell , W cr and F d .
8 . A cytometer as claimed in claim 7 , wherein the cell stiffness E is determined from the equation E = [ 6 D a + 3 ⁢ θ ⁢ D a D deform 2 - 8 π ⁢ ( 3 + v - 2 ⁢ v 2 ) ⁢ D Cell 2 + 2 ⁢ ( 1 - v 2 ) ⁢ D a 2 ( 1 - v 2 ) ⁢ ( D a 2 + 4 ⁢ D Cell 2 ) 3 / 2 ⁢ ( 1 + θ ⁢ D a 2 5 ⁢ D deform 2 ) ] ⁢ ψ ⁡ ( 1 - v 2 ) ⁢ F n 2 ⁢ ( D Cell - W cr ) where ⁢ D a = D cell 2 - W 2 + D deform - D Cell ⁢ ψ = ( 1 - ζ ) 2 ( 1 - ζ + ζ 2 / 3 ) ⁢ θ = 1 - ζ / 3 1 - ζ + ζ 2 / 3 ⁢ ζ = 1 - W cr D Cell ⁢ and ⁢ F n = F d 2 ⁢ μ where μ is the coefficient of friction of the sidewall and ν is the Poisson ratio which is set equal to 0.5.
9 . A cytometer as claimed in claim 6 , wherein the signal processor is further configured to compare each of the set of identified ripples to an expected ripple shape and to select the at least two ripples α, β which most closely resemble the expected ripple shape.
10 . A cytometer as claimed in claim 1 , wherein each channel portion is straight and of uniform width.
11 . A cytometer as claimed in claim 1 , further comprising a fluid source configured to provide a pressurised fluid to the channel portion of the first measurement section of each measurement channel.
12 . A method of cytometry comprising the steps of (a) providing a cytometer, the cytometer comprising a plurality of measurement sections, each measurement section comprising first and second sidewalls and a base extending therebetween, the first and second sidewalls and base defining a channel portion therebetween, the sidewalls and base extending from an entrance to an exit; an electrode group arranged on the opposite side of the base to the channel portion and part way between the entrance and exit, the electrode group comprising an upstream electrode, a downstream electrode and a center electrode, the electrodes being arranged such that as one travels from the entrance to the exit one passes over the upstream electrode, then the center electrode and then the downstream electrode; the channel portion having a width measured in a direction normal to the sidewalls; the measurement sections being connected together to form at least one measurement channel comprising a plurality of the channel portions connected together in series, with each channel portion of the measurement channel having a different width; and, at least one lock-in amplifier; the electrode groups being configured such that for each group the central electrode is connected to the excitation signal port of a lock-in amplifier and the upstream and downstream electrodes are connected to the voltage differential input ports of the lock-in amplifier; (b) identifying the set of ripples in the output which are produced by passage of a cell along different channel portions of a single measurement channel; (c) for at least two ripples α, β of the set of ripples which correspond to passage of the cell along channel portions ca and cp determining the peak-to-peak amplitudes V pp α and V pp β of the ripples; and, (d) solving the simultaneous equations V pp α =k ( D deform 2 ( D cell ,W cα )+ b ) V pp β =k ( D deform 2 ( D cell ,W cβ )+ b ) to determine a cell surface charge k and the undeformed diameter of the cell where b is a constant, W cα and W cβ are the widths of the channel portions cα and cβ and D deform (D cell ,W) is the diameter of a deformed cell of undeformed diameter in a channel of width and is related to D cell and W by the relation 4 3 ⁢ π ⁡ ( D cell 2 ) 3 = 2 ⁢ π ⁡ ( D deform 2 ) 2 ⁢ ( W 2 ) - 2 ⁢ π 3 ⁢ ( W 2 ) 3
13 . A method as claimed in claim 12 , further comprising the steps of (e) selecting a ripple r from the set of ripples and for ripple r divide a predetermined constant C by the peak-to-peak time t pp of the ripple r to determine the speed of the cell which caused the ripple; (f) determining driving force F d on the cell in the channel portion from a model for channel portion cr of the driving force as a function of cell speed and D cell ; and (g) determining cell stiffness E from D cell , W cr and F d .
14 . A method as claimed in claim 13 , wherein the cell stiffness E is determined from the equation E = [ 6 D a + 3 ⁢ θ ⁢ D a D deform 2 - 8 π ⁢ ( 3 + v - 2 ⁢ v 2 ) ⁢ D Cell 2 + 2 ⁢ ( 1 - v 2 ) ⁢ D a 2 ( 1 - v 2 ) ⁢ ( D a 2 + 4 ⁢ D cell 2 ) 3 / 2 ⁢ ( 1 + θ ⁢ D a 2 5 ⁢ D deform 2 ) ] ⁢ ψ ⁡ ( 1 - v 2 ) ⁢ F n 2 ⁢ ( D Cell - W cr ) where ⁢ D a = D cell 2 - W 2 + D deform - D Cell ⁢ ψ = ( 1 - ζ ) 2 ( 1 - ζ + ζ 2 / 3 ) ⁢ θ = 1 - ζ / 3 1 - ζ + ζ 2 / 3 ⁢ ζ = 1 - W cr D Cell ⁢ and ⁢ F n = F d 2 ⁢ μ where μ is the coefficient of friction of the sidewall and ν is the Poisson ratio which is set equal to 0.5.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority to U.S. Provisional Application No. 63/304,477 filed with the United States Patent and Trademark Office on Jan. 28, 2022 and entitled “HIGH-THROUGHPUT ELECTRICAL CELL CYTOMETRY FOR WHOLE-CELL BIOPHYSICAL PROPERTIES,” which is incorporated herein by reference in its entirety for all purposes. TECHNICAL FIELD OF THE INVENTION The present invention relates to a cytometer. More particularly, but not exclusively, the present invention relates to a cytometer comprising a plurality of measurement sections, each measurement section comprising a channel portion defined by a base and sidewalls and an electrode group arranged on the opposite side of the base to the channel portion, the measurement sections being connected together to form at least one measurement channel comprising a plurality of channel portions connected in series, the cytometer further comprising at least one lock-in amplifier, the electrode groups being connected to the at least one lock-in amplifier. The present invention also relates to a method of cytometry. More particularly, but not exclusively, the present invention relates to a method of cytometry comprising the steps of providing a cytometer according to the invention and determining the undeformed diameter and surface charge of cells passing along the channel portions from the output of the at least one lock-in amplifier. BACKGROUND Cell physical properties including size, shape, elastic modulus, cytoplasmic viscosity and electric permittivity, are determined by the intracellular structures and molecular compositions. Cell physical properties can be label free and non-invasive biomarkers, as they vary upon some disease and the underlying pathological molecular alterations in the involved cells. For example, cell elastic modulus is well known to be related to malignancy and invasiveness of tumor cells and the immunological state of immune cells. It has also been demonstrated that electric permittivity of cells reflects intercellular communication, cell adhesion and nutrient absorption. Mechanical and electrical properties exhibit cell phenotypes in different aspects, eg cell stiffness for cell motility and alpha dispersion for charges membrane molecules. Multiparametric phenotyping of individual cells for both mechanical and electrical properties can achieve a more promising prognostic/diagnostic strategy. Ideally these properties should not correlate among others, and each reflect a unique physical aspect. Physical cell phenotyping techniques are known, yet the concerned cell properties are ‘monotonic’ ie either mechanical or electrical. This limits the phenotyping depth and the applicability in disease prognosis/diagnosis and cell classification. Known techniques of mechanical cell phenotyping include deformability cytometry which is based on microscopic imaging of cell deformation upon flow stresses in microchannels. Known techniques of electrical cell phenotyping include impedance cytometry. The present invention seeks to overcome the problems of the prior art. SUMMARY OF INVENTION Accordingly, in a first aspect the present invention provides a cytometer comprising a plurality of measurement sections, each measurement section comprising first and second sidewalls and a base extending therebetween, the first and second sidewalls and base defining a channel portion therebetween, the sidewalls and base extending from an entrance to an exit;an electrode group arranged on the opposite side of the base to the channel portion and part way between the entrance and exit, the electrode group comprising an upstream electrode, a downstream electrode and a center electrode, the electrodes being arranged such that as one travels from the entrance to the exit one passes over the upstream electrode, then the center electrode and then the downstream electrode;the channel portion having a width measured in a direction normal to the sidewalls; the measurement sections being connected together to form at least one measurement channel comprising a plurality of the channel portions connected together in series, with each channel portion of the measurement channel having a different width; and,at least one lock-in amplifier;the electrode groups being configured such that for each group the central electrode is connected to the excitation signal port of a lock-in amplifier and the upstream and downstream electrodes are connected to the voltage differential input ports of the lock-in amplifier. The cytometer according to the invention enables measurement of undeformed cell diameter, cell surface charge and cell stiffness from the output of the at least one lock-in amplifier. Preferably the at least one measurement channel comprises first and second channel portions only. Alternatively, the at least one measurement channel comprises at least three channel portions. Preferably the measurement sections are connected together to form a