JPWO2021146350A5 - - Google Patents

Description

The two false negatives in the data set barely meet the test threshold (>33 Ct) and appear to be random rather than time-related. Due to its high sensitivity, the SARS-CoV-2 Ag test (LOD32TCID50/ml) correctly identifies all positive patients with Ct<33. Based on the reported LOD values of other SARS-CoV-2 antigen tests, it appears that those tests may not be able to identify Ct>30. Based on this (illustrated) data set, a test that fails to discriminate Ct>30 translates into a comparative sensitivity of approximately 80% (51/65).
The claims as filed are as follows.
<Claim 1>
(a) introducing a sample liquid into a microchannel of a microfluidic device, the sample liquid occupying a first portion of the microchannel, and a second portion of the microchannel adjacent to the first portion; , the sample liquid and the gas forming a liquid-gas interface therebetween;
(b) inducing pressure oscillations in the sample liquid by repeatedly changing the pressure of the gas in the second portion of the microchannel;
A method characterized by comprising:
<Claim 2>
The step of introducing the sample liquid into the microchannel includes applying the sample liquid to a sample introduction port of the microfluidic device,
the first portion of the microchannel is downstream of the sample introduction port (i.e. distal within the channel or network to the application zone);
the second portion of the microchannel is downstream of the first portion of the microchannel
The method according to claim 1, characterized in that:
<Claim 3>
The step of repeatedly varying the pressure of the gas in the second portion of the microchannel is performed at a frequency of at least about 10 Hz, a frequency of at least about 25 Hz, a frequency of at least about 100 Hz, a frequency of at least about 250 Hz, a frequency of at least about 500 Hz. , at a frequency of at least about 700 Hz, at a frequency of at least about 750 Hz, or at a frequency of at least about 1000 Hz.
The method according to claim 1 or 2, characterized in that:
<Claim 4>
The step of repeatedly changing the pressure of the gas in the second portion of the microchannel is performed at a frequency below the acoustic frequency, such as a frequency below about 2000 Hz, a frequency below about 1500 Hz, a frequency below about 1250 Hz, a frequency below about 1000 Hz. , at a frequency of about 900 Hz or less, or at a frequency of about 800 Hz or less
The method according to any one of claims 1 to 3, characterized in that:
<Claim 5>
The step of repeatedly changing the pressure of the gas in the second portion of the microchannel includes vibrating walls of the second portion of the microchannel.
The method according to any one of claims 1 to 4, characterized in that:
<Claim 6>
The step of vibrating the wall of the second portion of the microchannel causes the wall to vibrate over the entire peak-to-peak distance at a frequency of the step of changing the pressure of the gas in the second portion of the microchannel. including the step of vibrating the
The total peak-to-peak distance is measured along an axis perpendicular to a plane defined by the microfluidic device and is about 75 μm or less, about 65 μm or less, about 50 μm or less, about 40 μm or less, about 25 μm or less, about 20 μm or less, about 15 μm or less, about 10 μm or less, about 8 μm or less, about 7 μm or less, or about 6 μm or less
6. The method according to claim 5, characterized in that:
<Claim 7>
Said step of vibrating said wall of said second portion of said microchannel vibrates said wall over a full peak-to-peak distance at a frequency of said step of changing the pressure of said gas in said second portion of said microchannel. including the step of vibrating the
The total peak-to-peak distance is measured along an axis perpendicular to a plane defined by the microfluidic device and is at least about 1 μm, at least about 2 μm, at least about 2.5 μm, at least about 3 μm, at least about 4 μm. , at least about 5 μm, at least about 10 μm, at least about 15 μm, or at least about 20 μm.
6. The method according to claim 5, characterized in that:
<Claim 8>
The step of vibrating the wall of the second portion of the microchannel is carried out by contacting an outer surface of the wall of the second portion of the microchannel with a mechanical member.
The method according to any one of claims 5 to 7, characterized in that:
<Claim 9>
over a distance measured along an axis perpendicular to a plane defined by the microfluidic device that is approximately the same distance as traveled by the wall of the second portion of the microchannel. , vibrating the mechanical member;
9. The method according to claim 8, comprising:
<Claim 10>
The step of contacting the outer surface of the wall of the second portion of the microchannel with a mechanical member includes contacting the wall of the second portion of the microchannel with the mechanical member by about 12 mm.²Approximately 10mm below²Approximately 8mm below²Approximately 6mm below²Less than or about 5mm²The following includes the step of contacting over the entire area of
The method according to claim 8 or 9, characterized in that.
<Claim 11>
The step of contacting an outer surface of the wall of the second portion of the microchannel with a mechanical member includes contacting the wall of the second portion of the microchannel with the mechanical member by at least about 1 mm.², at least about 2mm², at least about 3mm², at least about 4mm², or at least about 5mm², including the step of contacting over the entire area of
The method according to any one of claims 8 to 10, characterized in that:
<Claim 12>
The step of contacting the outer surface of the wall of the second portion of the microchannel with a mechanical member may include contacting the outer surface of the wall of the second portion of the microchannel with the mechanical member and the outer surface of the wall of the second portion of the microchannel at the contact location. contacting over a distance corresponding to at least about 10%, at least about 15%, at least about 20%, or at least about 25% of the width of the second portion.
A method according to any one of claims 8 to 11, characterized in that:
<Claim 13>
The step of contacting the outer surface of the wall of the second portion of the microchannel with a mechanical member may include contacting the outer surface of the wall of the second portion of the microchannel with the mechanical member and the outer surface of the wall of the second portion of the microchannel at the contact location. contacting over a distance corresponding to about 35% or less, about 30% or less, or about 25% or less of the width of the second portion.
A method according to any one of claims 8 to 12, characterized in that:
<Claim 14>
the width of the second portion of the microchannel at the location of contact with the mechanical member is at least about 1.25 times greater than the width of the first portion of the microchannel occupied by the liquid sample; at least about 1.5 times larger, or at least about 2 times larger
A method according to any one of claims 8 to 13, characterized in that:
<Claim 15>
The step of vibrating the mechanical member includes actuating, e.g. piezoelectrically, the mechanical member in contact with the outer surface of the wall of the second portion of the microchannel.
15. The method according to any one of claims 8 to 14.
<Claim 16>
the mechanical member is connected to an actuator, e.g. a piezoelectric actuator, via a laterally extending arm, e.g. a piezoelectric bender;
the actuator is laterally offset from the first and second portions of the microchannel
16. The method according to claim 15, characterized in that:
<Claim 17>
The actuator is laterally offset from the area contacted by the mechanical member by a distance of at least about 1 cm, at least about 1.5 cm, or at least about 2 cm.
17. The method according to claim 16, characterized in that:
<Claim 18>
compressing the walls of the second portion of the microchannel before introducing the sample liquid into the microchannel;
maintaining compression of the wall of the microchannel while introducing the sample liquid into the microchannel;
18. The method according to any one of claims 1 to 17, further comprising:
<Claim 19>
an interior of the second portion of the microchannel includes first and second spaced apart electrical contacts;
The compressing step includes compressing the wall of the second portion of the microchannel until an electrical signal is received indicating that the first and second electrical contacts are in electrical continuity.
19. A method according to claim 18, characterized in that.
<Claim 20>
After receiving the electrical signal and before introducing the sample liquid into the microchannel, the microchannel reducing the compression of the wall of the second portion of the channel;
20. The method of claim 19, further comprising:
<Claim 21>
said compressing step comprises compressing said wall of said second portion of said microchannel by a maximum distance D measured along an axis perpendicular to a plane defined by said microfluidic device;
The method includes at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of the compression relative to the distance D before introducing the sample liquid into the microchannel. The process of maintaining % or essentially all
21. The method according to any one of claims 18 to 20, further comprising:
<Claim 22>
The step of compressing the second portion of the microchannel increases the internal height of the second portion of the microchannel measured along an axis perpendicular to a plane defined by the microfluidic device. at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%.
22. A method according to any one of claims 18 to 21, characterized in that:
<Claim 23>
The step of compressing the second portion of the microchannel increases the internal height of the second portion of the microchannel measured along an axis perpendicular to a plane defined by the microfluidic device. reducing by at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 75 μm, at least about 85 μm, or at least about 90 μm.
22. A method according to any one of claims 18 to 21, characterized in that:
<Claim 24>
The overall internal height of the second portion of the microchannel, measured along an axis perpendicular to the plane defined by the microfluidic device, is between about 50 and 200 μm before the compacting step. , between about 75 and 150 μm, between about 90 and 130 μm, or about 110 μm
24. A method according to any one of claims 18 to 23, characterized in that:
<Claim 25>
The step of compressing the walls of the second portion of the microchannel forces a portion of the gas from the second portion of the microchannel into at least the first portion of the microchannel.
25. A method according to any one of claims 18 to 24.
<Claim 26>
before the compressing step, the outer surface of the wall of the second portion of the microchannel is substantially planar;
After the compressing step, the outer surface of the wall of the second portion of the microchannel is concave.
26. A method according to any one of claims 18 to 25.
<Claim 27>
upon introduction of the sample liquid, the sample liquid passes through at least a portion of the microchannel by capillary action;
(i) the leading liquid-gas interface of the sample liquid is located within the channel upstream of at least the second portion of the microchannel (i.e. proximal within the channel or network to the application zone); until reaching a first capillary stop located; and/or
(ii) until the gas pressure downstream of the leading liquid-gas interface is high enough to stop capillary flow of the sample liquid further downstream;
flowing
27. A method according to any one of claims 18 to 26.
<Claim 28>
after the downstream flow of the sample liquid has ceased, reducing the gas pressure at the leading liquid-gas interface of the sample liquid by reducing the compression applied to the second portion of the microchannel; , thereby causing the sample liquid to move a further distance along the microchannel towards the second portion of the microchannel.
28. The method of claim 27, further comprising:
<Claim 29>
at least about 10 μm/s, at least about 20 μm/s, at least about 50 μm/s, at least about 400 μm/s from the leading liquid-gas interface of the sample liquid toward the second portion of the microchannel. s, at least about 600 μm/s, at least about 750 μm/s, at least about 1000 μm/s, at least about 1250 μm/s, or at least about 1500 μm/s.
reducing the compression applied to the second portion of the microchannel at a rate sufficient to
29. The method of claim 28, further comprising:
<Claim 30>
about 2000 μm/s or less, about 1900 μm/s or less, about 1800 μm/s or less, about 1500 μm/s from the leading liquid-gas interface of the sample liquid toward the second portion of the microchannel. Below, about 1250 μm/s or less, about 1000 μm/s or less, about 750 μm/s or less, about 500 μm/s or less, about 250 μm/s or less, about 150 μm/s or less, about 100 μm/s or less, or about 75 μm/s Move at the following speed,
reducing the compression applied to the second portion of the microchannel at a rate sufficient to
30. The method according to claim 28 or 29, further comprising:
<Claim 31>
the further distance along the microchannel between the leading liquid-gas interface of the sample liquid when first stopped and the point of maximum compression of the second portion of the microchannel; between about 10% and 60%, between about 20% and 50%, between about 25% and 40%, about 25%, about 35%, or about 50 of the total distance
31. A method according to any one of claims 28 to 30.
<Claim 32>
Said further distance is
at least about 1 mm, at least about 2 mm, at least about 3 mm, at least about 4 mm, or at least about 5 mm,
is
32. A method according to any one of claims 28 to 31, characterized in that:
<Claim 33>
The further distance is
It is about 10 mm or less, about 9 mm or less, about 8 mm or less, about 7 mm or less, about 6 mm or less, or about 5 mm or less
33. A method according to any one of claims 28 to 32.
<Claim 34>
The additional distance may be such that the sample liquid is at least about 100 nL, at least about 200 nL, at least about 300 nL, or at least about 400 nL gas in front of the leading liquid-gas interface in the microchannel. replace the volume of
34. A method according to any one of claims 28 to 33, characterized in that:
<Claim 35>
The additional distance may be such that the sample liquid is no more than about 1000 nL, no more than about 900 nL, no more than about 800 nL, no more than about 700 nL, no more than about 600 nL, or no more than about 500 nL of the leading liquid in the microchannel. Displace the volume of gas in front of the gas interface
35. A method according to any one of claims 28 to 34.
<Claim 36>
the microchannel has a first reagent zone containing one or more first reagents deposited therein;
the reagent zone is located between the leading liquid-gas interface of the sample liquid when first stopped and the point of maximum compression of the second portion of the microchannel;
The further distance is sufficient to cause the leading liquid-gas interface of the sample liquid to traverse the entire first reagent zone.
36. A method according to any one of claims 28 to 35.
<Claim 37>
The step of reducing the compression applied to the second portion of the microchannel is performed simultaneously with the application of an energy pulse.
37. A method according to any one of claims 28 to 36.
<Claim 38>
The step of changing the pressure of the gas in the second portion of the microchannel induces mixing.
38. A method according to claim 37, characterized in that.
<Claim 39>
after the leading liquid-gas interface of the sample liquid has moved a predetermined further distance along the microchannel toward the second portion of the microchannel. stopping said step of reducing compaction;
further comprising;
The sample liquid is caused by capillary action to
(i) a second capillary in the channel, wherein the leading liquid-gas interface of the sample liquid is located downstream of the first capillary stop and upstream of at least the second portion of the microchannel; until a stop is reached, and/or
(ii) until the gas pressure downstream of the leading liquid-gas interface is high enough to stop capillary flow of the sample liquid further downstream;
flowing
39. A method according to any one of claims 28 to 38.
<Claim 40>
the reagent is mobilizable by the sample liquid
40. A method according to any one of claims 37 to 39, characterized in that.
<Claim 41>
further reducing the compression applied to the second portion of the microchannel after downstream flow of the sample liquid has ceased following cessation of the step of reducing compression of the second portion of the microchannel; The gas pressure at the leading liquid-gas interface of the sample liquid is again reduced by causing the sample liquid to move further along the microchannel toward the second portion of the microchannel. The process of moving again by a distance
41. The method according to claim 39 or 40, further comprising:
<Claim 42>
at least about 400 μm/s, at least about 600 μm/s, at least about 750 μm/s, at least about 1000 μm/s from the leading liquid-gas interface of the sample liquid toward the second portion of the microchannel. s, at least about 1250 μm/s, or at least about 1500 μm/s.
reducing the compression applied to the second portion of the microchannel at a rate sufficient to
42. The method of claim 41, further comprising:
<Claim 43>
The leading liquid-gas interface of the sample liquid toward the second portion of the microchannel is about 2000 μm/s or less, about 1900 μm/s or less, about 1800 μm/s or less, or about 1700 μm/s. Move at a speed of /s or less,
reducing the compression applied to the second portion of the microchannel at a rate sufficient to
43. The method according to claim 41 or 42, further comprising:
<Claim 44>
The further distance is along the microchannel between the leading liquid-gas interface of the sample liquid when first stopped and the point of maximum compression of the second portion of the microchannel. between about 10% and 60%, between about 20% and 50%, between about 25% and 40%, about 25%, about 35%, or about 50 of the total distance
44. A method according to any one of claims 41 to 43, characterized in that:
<Claim 45>
The further distance is
at least about 1 mm, at least about 2 mm, at least about 3 mm, at least about 4 mm, or at least about 5 mm,
is
45. A method according to any one of claims 41 to 44.
<Claim 46>
Said further distance is
It is about 10 mm or less, about 9 mm or less, about 8 mm or less, about 7 mm or less, about 6 mm or less, or about 5 mm or less.
46. A method according to any one of claims 41 to 45.
<Claim 47>
The additional distance may be such that the sample liquid is at least about 100 nL, at least about 200 nL, at least about 300 nL, or at least about 400 nL gas in front of the leading liquid-gas interface in the microchannel. replace the volume of
47. A method according to any one of claims 41 to 46.
<Claim 48>
The additional distance may be such that the sample liquid is no more than about 1000 nL, no more than about 900 nL, no more than about 800 nL, no more than about 700 nL, no more than about 600 nL, or no more than about 500 nL of the leading liquid in the microchannel. Displace the volume of gas in front of the gas interface
48. A method according to any one of claims 41 to 47.
<Claim 49>
the microchannel has a second reagent zone containing one or more second reagents deposited therein;
the second reagent zone is located between the first reagent zone and the point of maximum compression of the second portion of the microchannel;
The further distance is sufficient to cause the leading liquid-gas interface of the sample liquid to traverse the entire second reagent zone.
49. A method according to any one of claims 41 to 48.
<Claim 50>
the second portion of the microchannel after the leading liquid-gas interface of the sample liquid has moved again a predetermined further distance along the microchannel toward the second portion of the microchannel; stopping the step of further reducing the compression of the
further comprising;
The sample liquid is caused by capillary action to
(i) a second capillary in the channel, wherein the leading liquid-gas interface of the sample liquid is located downstream of the first capillary stop and upstream of at least the second portion of the microchannel; until a stop is reached, and/or
(ii) until the gas pressure downstream of the leading liquid-gas interface is high enough to stop capillary flow of the sample liquid further downstream;
flowing
50. A method according to any one of claims 41 to 49.
<Claim 51>
performing said step of repeatedly varying the pressure of said gas in said second portion of said microchannel while simultaneously performing said step of reducing said compression applied to said second portion of said microchannel;
51. The method according to any one of claims 43 to 50, further comprising:
<Claim 52>
The step of changing the pressure of the gas in the second portion of the microchannel induces mixing.
52. The method of claim 51.
<Claim 53>
The microchannel is in gas communication with the ambient atmosphere upstream of the second portion of the microchannel and sealed to the ambient atmosphere downstream of the second portion of the microchannel; wherein compression of the second portion of the microchannel displaces gas from the second portion of the microchannel toward the first portion of the microchannel.
53. A method according to any one of claims 1 to 52, characterized in that:
<Claim 54>
maintaining at least about 50%, at least about 65%, at least about 75%, at least about 85%, or at least about 90% of the compression prior to inducing pressure oscillations in the sample liquid;
54. A method according to any one of claims 18 to 53, further comprising:
<Claim 55>
The liquid-gas interface is oriented substantially perpendicular to the longitudinal axis of the microchannel.
55. A method according to any one of claims 1 to 54, characterized in that:
<Claim 56>
the first and second portions of the microchannel are sequentially positioned along a longitudinal axis of the microchannel;
56. A method according to any one of claims 1 to 55.
<Claim 57>
the liquid-gas interface is oriented along a substantially vertical axis;
The longitudinal axis of the microchannel is oriented along a generally horizontal axis.
57. A method according to any one of claims 1 to 56, characterized in that:
<Claim 58>
the liquid along the microchannel over a distance greater than the amplitude of the oscillations along the microchannel while simultaneously performing the steps of repeatedly varying the pressure of the gas in the second portion of the microchannel; Process of moving the average position of the gas interface in parallel
58. A method according to any one of claims 1 to 57, further comprising:
<Claim 59>
The sample liquid includes a fluorescent tag bound to magnetic particles by an immunological link, and a fluorescent tag that does not contain magnetic particles,
The method is
applying a magnetic field to the first portion of the microchannel while simultaneously performing the steps of repeatedly changing the pressure of the gas in the second portion of the microchannel;
59. A method according to any one of claims 1 to 58, further comprising:
<Claim 60>
The axis of the magnetic field is oriented substantially parallel to an axis of symmetry defined by the liquid-gas interface.
60. The method of claim 59.
<Claim 61>
translating the position of the liquid-gas interface along the longitudinal axis of the microchannel;
further comprising;
The axis of the magnetic field is oriented substantially perpendicular to the longitudinal axis of the microchannel.
61. A method according to claim 59 or 60, characterized in that.
<Claim 62>
the first portion of the microchannel has a plurality of liquid-gas interfaces of sample liquid spaced from each other along a longitudinal axis of the first portion of the microchannel;
Iteratively changing the pressure of the gas in the second portion of the microchannel includes oscillating the position of the plurality of interfaces with respect to the longitudinal axis of the microchannel.
59. A method according to any one of claims 1 to 58, characterized in that:
<Claim 63>
Oscillations in the position of each interface occur along an axis substantially perpendicular to the longitudinal axis of the first portion of the microchannel.
63. The method of claim 62.
<Claim 64>
A method of moving a sample liquid within a microfluidic device, the method comprising:
(a) compressing a portion of the wall of the microchannel of the microfluidic device;
(b) introducing a sample liquid into the microchannel, the liquid traveling only part way along the microchannel towards the compressed wall of the microchannel;
(c) directing the sample liquid along the microchannel and toward the compressed wall of the microchannel by at least partially reducing the compression of the wall and vibrating the compressed wall; and further moving the
A method characterized by comprising:
<Claim 65>
a step of simultaneously performing the step of reducing the compression and the step of vibrating the wall;
65. The method of claim 64, comprising:
<Claim 66>
The step of compressing the walls includes reducing the height of the microchannel by at least about 50 μm, at least about 60 μm, or at least about 70 μm.
66. A method according to claim 64 or 65, characterized in that.
<Claim 67>
The step of vibrating the wall vibrates the wall by a distance of no more than about 10 μm, no more than about 7.5 μm, or no more than about 5 μm, measured along a dimension corresponding to the height of the microchannel. including process
67. A method according to any one of claims 64 to 66.
<Claim 68>
The step of vibrating the wall includes vibrating the wall a distance of at least about 1 μm, at least about 2 μm, or at least about 2.5 μm.
68. A method according to any one of claims 64 to 67.
<Claim 69>
(a) providing a capillary tube, the capillary tube having a capillary channel defining a longitudinal axis and containing a liquid and a gas;
(b) vibrating the pressure of the gas;
A method comprising:
the liquid and the gas are disposed within respective successive first and second portions of the capillary channel along the longitudinal axis;
The liquid and the gas form a gas-liquid interface between them.
A method characterized by:
<Claim 70>
the capillary defines a plurality of cavities spaced from each other along the longitudinal axis of the first portion of the capillary channel;
each cavity contains a gas disposed therein;
the gas and the liquid within each cavity forming a gas-liquid interface therebetween;
70. The method of claim 69.
<Claim 71>
the wall is an exterior wall
69. A method according to any one of claims 5 to 68.
<Claim 72>
first and second substrates fixed relative to each other, both having a generally planar extent and at least partially defining a microfluidic channel network;
reagent and
Equipped with
the first substrate defines an inner surface above or below the microchannels of the microfluidic channel network;
the second substrate defines at least one of two opposing sidewalls of the microchannel;
a first portion of the reagent is disposed within the microchannel on the upper or lower inner surface of the microchannel between the two opposing sidewalls of the microchannel;
A second portion of the reagent is located between the first and second substrates along an axis substantially perpendicular to the generally planar extent of the first and second substrates and in the microchannel. placed on the outside
A microfluidic device characterized by:
<Claim 73>
a second substrate fixed to the second substrate and having a generally planar extent with the first and second substrates and at least partially defining the microfluidic channel network with the first and second substrates; 3 board
further comprising;
The third substrate defines the other of the upper or lower inner surfaces of the microchannel.
73. The microfluidic device of claim 72.
<Claim 74>
The reagents include lysis reagents, buffer reagents, detectably labeled reagents (e.g., fluorescently labeled reagents), reagents configured to specifically bind to the target to be detected, magnetically selected from the group consisting of labeled reagents or combinations thereof;
74. The microfluidic device according to claim 72 or 73.
<Claim 75>
The reagent is in a non-liquid state, for example in a dry or lyophilized state.
75. The microfluidic device according to any one of claims 72 to 74.
<Claim 76>
During use of the microfluidic device,
the first portion of the reagent within the microchannel is solubilized by a sample liquid;
Substantially all of the second portion of the reagent outside the microchannel remains solubilized by the sample liquid, and/or the substantially planar portion of the first and second substrates remains solubilized by the sample liquid. between the first and second substrates and outside the microchannel along the axis substantially perpendicular to the extent of the microchannel;
76. The microfluidic device according to any one of claims 72 to 75.
<Claim 77>
At least one, such as at least two or all three of the first, second and third substrates are substantially perpendicular to the substantially planar extent of the first and second substrates. Consisting of multiple layers along the axis
77. The microfluidic device according to any one of claims 73 to 76.
<Claim 78>
At least one, such as at least two or all three of the first, second and third substrates are substantially parallel to the substantially planar extent of the first and second substrates. Consists of two or more separate substrates arranged along an axis
78. A microfluidic device according to claims 73 to 77.
<Claim 79>
The second substrate includes an adhesive layer that secures the first and second substrates together and secures the second and third substrates together.
79. A microfluidic device according to any one of claims 73 to 78.
<Claim 80>
The second substrate defines two opposing sidewalls of the microchannel.
80. A microfluidic device according to any one of claims 72 to 79.
<Claim 81>
the microchannel defines a longitudinal axis;
at least one sidewall, e.g. both sidewalls, of the microchannel define a plurality of cavities;
Each of the plurality of cavities has a longitudinal axis oriented substantially perpendicular to the longitudinal axis at the location of the plurality of cavities.
81. The microfluidic device according to any one of claims 72 to 80.
<Claim 82>
The second portion of the reagent comprises:
(i) between the first and second substrates and outside the microchannel along an axis substantially perpendicular to the generally planar extent of the first and second substrates;
(ii) between adjacent cavities along an axis substantially parallel to the longitudinal axis of the channel at a location between the adjacent cavities;
Contains arranged reagents
82. The microfluidic device of claim 81.
<Claim 83>
The second substrate defines two opposing sidewalls of the microchannel.
83. A microfluidic device according to any one of claims 72 to 82.
<Claim 84>
A microfluidic device,
a microfluidic channel network comprising a microchannel configured to receive a liquid and a mechanically manipulated zone in fluid communication with the microchannel;
a first electrode positioned to contact a liquid within the microfluidic channel network at a first location;
a first electrically conductive lead extending from the first electrode to a first electrical contact located on the microfluidic device outside of the microfluidic channel network;
a second electrode positioned to contact a liquid within the microfluidic network at a second location spaced apart from the first location;
a second electrically conductive lead extending from the second electrode to a second electrical contact located on the microfluidic device outside of the microfluidic channel network and spaced apart from the first electrical contact; ,
Equipped with
the mechanical manipulation zone includes a first manipulation part and a second manipulation part;
mechanical manipulation of one of the first and second manipulating parts induces movement of liquid, if present, in the microchannel relative to the other of the first and second manipulating parts;
the first electrically conductive lead includes a first lead portion disposed within or adjacent the mechanical manipulation zone;
the second electrically conductive lead includes a second lead portion disposed within or adjacent the mechanical manipulation zone;
the first and second electrodes are each configured to perform a respective liquid sensing function or target detection function at respective first and second positions;
Mechanical manipulation of one of the first and second manipulating parts brings the first and second leads into electrical communication with each other with respect to the other of the first and second manipulating parts, or cutting off electrical continuity between the first and second lead wires;
A microfluidic device characterized by:
<Claim 85>
The mechanical manipulation zone is a gas bladder in fluid communication with the microchannel.
85. The microfluidic device of claim 84.
<Claim 86>
the first operating part is a first wall of the gas bladder;
the second operating part is a second wall of the gas bladder;
The first and second walls are arranged opposite to each other.
86. The microfluidic device of claim 85.
<Claim 87>
The first and second manipulating portions are arranged such that compression of the mechanical manipulating zone brings the first and second leads into electrical communication with each other.
87. The microfluidic device according to claim 85 or 86.
<Claim 88>
the first and second operating portions are each disposed on an inner surface of one of the first and second walls of the mechanical operating zone;
The interior of the other of the first and second walls includes an electrically conductive surface configured to bring the first and second leads into electrical communication with each other upon compression of the mechanical manipulation zone.
88. The microfluidic device of claim 87.
<Claim 89>
The conductive surface is a surface of a conductive bridge member fixed within the other of the first and second walls.
89. The microfluidic device of claim 88.
<Claim 90>
A method for detecting anti-coronavirus spike protein antibodies in a sample from a subject, the method comprising:
applying said sample to a serological assay comprising first and second reagents;
Equipped with
the first reagent comprises a receptor binding domain (RBD) of the coronavirus spike protein or a fragment thereof, and is conjugated or configured to bind a detectable label or capture agent;
the second reagent is bound or configured to bind a detectable label or capture agent;
the first reagent and the second reagent bind to an anti-coronavirus spike protein antibody to form a complex comprising the first reagent, the anti-coronavirus spike protein antibody, and the second reagent;
Formation of said complex indicates the presence of said anti-coronavirus spike protein antibody within said sample.
A method characterized by:
<Claim 91>
The second reagent contains the S1 subunit of the coronavirus spike protein or a fragment thereof.
91. The method of claim 90.
<Claim 92>
the amino acid sequence of the RBD is at least 75%, 80%, 85%, 90%, 95%, 96% of amino acids 319-541 of the SARS-CoV-2 spike protein (SEQ ID NO: 1); have 97%, 98%, 99%, 99.5, or 100% sequence identity
92. A method according to claim 90 or 91, characterized in that.
<Claim 93>
The RBD and/or S1 subunit of the coronavirus spike protein, or a fragment thereof further comprises an Fc region.
93. A method according to any one of claims 90 to 92.
<Claim 94>
The first reagent is bound or configured to bind a detectable label.
94. A method according to any one of claims 90 to 93.
<Claim 95>
The first reagent is bound to or configured to bind to a capture agent.
95. A method according to any one of claims 90 to 94.
<Claim 96>
The second reagent is bound or configured to bind a detectable label.
96. The method according to any one of claims 90 to 93 and 95.
<Claim 97>
The second reagent is bound or configured to bind to a capture agent.
97. A method according to any one of claims 90 to 94, 96.
<Claim 98>
The detectable label includes fluorescent particles, such as fluorescent latex beads.
95. A method according to any one of claims 90 to 94.
<Claim 99>
The capture agent includes biotin, avidin, streptavidin, and/or magnetic beads.
99. A method according to any one of claims 1 to 98.
<Claim 100>
The method is carried out in a microfluidic device according to any of claims 72 to 89.
100. A method according to any one of claims 90 to 99.
<Claim 101>
The coronavirus is SARS-CoV-2
101. A method according to any one of claims 90 to 100.
<Claim 102>
The sample includes blood, serum, or plasma.
102. A method according to any one of claims 90 to 101.
<Claim 103>
Before applying the sample to the binding assay, the sample is contacted with latex particles.
103. A method according to any one of claims 90 to 102.
<Claim 104>
Before applying the sample to the binding assay, the sample is contacted with a buffer containing a salt solution.
104. A method according to any one of claims 90 to 103.
<Claim 105>
When applying said sample to said binding assay, the presence of said anti-coronavirus spike protein antibodies is detected.
105. A method according to any one of claims 90 to 104.
<Claim 106>
The reagent includes a capture agent or a detectable label.
106. A method according to any one of claims 100 to 105.
<Claim 107>
The microchannel includes a dried first reagent and a second reagent therein,
the first reagent comprises a receptor binding domain (RBD) of the coronavirus spike protein or a fragment thereof, and is conjugated or configured to bind a detectable label or capture agent;
the second reagent is bound or configured to bind a detectable label or capture agent;
The first reagent and the second reagent, when solubilized by the sample, include the first reagent, an anti-coronavirus spike protein antibody if present in the sample, and the second reagent. form a complex
90. A microfluidic device according to any one of claims 72 to 89.
<Claim 108>
the amino acid sequence of the RBD is at least 75%, 80%, 85%, 90%, 95%, 96% of amino acids 319-541 of the SARS-CoV-2 spike protein (SEQ ID NO: 1); have 97%, 98%, 99%, 99.5, or 100% sequence identity
108. The microfluidic device of claim 107.
<Claim 109>
The RBD or S1 subunit of the coronavirus spike protein, or a fragment thereof further comprises an Fc region.
109. The microfluidic device according to claim 107 or 108.
<Claim 110>
The first reagent is bound or configured to bind a detectable label.
110. The microfluidic device according to any one of claims 107 to 109.
<Claim 111>
The first reagent is bound to or configured to bind to a capture agent.
111. The microfluidic device according to any one of claims 107 to 110.
<Claim 112>
The second reagent is bound or configured to bind a detectable label.
112. The microfluidic device according to any one of claims 107 to 109 and 111.
<Claim 113>
The second reagent is bound or configured to bind to a capture agent.
113. The microfluidic device according to any one of claims 107 to 110 and 112.
<Claim 114>
The detectable label includes fluorescent particles, such as fluorescent latex beads.
114. The microfluidic device according to any one of claims 107 to 113.
<Claim 115>
The capture agent includes magnetic beads.
115. The microfluidic device according to any one of claims 101 to 114.
<Claim 116>
The coronavirus is SARS-CoV-2
116. The microfluidic device according to any one of claims 107 to 115.
<Claim 117>
The sample includes blood, serum, or plasma.
117. The microfluidic device according to any one of claims 107 to 116.
<Claim 118>
A microfluidic device for detecting anti-coronavirus spike protein antibodies in a sample from a subject, the device comprising:
a first microchannel containing a dried first reagent and a second reagent therein;
a second microchannel containing a dried first reagent and a second reagent therein;
Equipped with
the first and second reagents in the first microchannel each include the S1 subunit of coronavirus spike glycoprotein;
the first reagent in the second microchannel comprises the S1 subunit of the coronavirus spike glycoprotein;
the second reagent in the second microchannel comprises a receptor binding domain (RBD) of the coronavirus spike protein;
each of the first reagents is bound or configured to bind a detectable label or capture agent;
each of the second reagents is bound or configured to bind a detectable label or capture agent;
Each of the first reagent and the second reagent, when solubilized by the sample, forms a complex comprising the first reagent, the anti-coronavirus spike protein antibody, and the second reagent.
A microfluidic device characterized by:
<Claim 119>
a third microfluidic channel containing the same first and second reagents as the first and second reagents in the second microchannel;
120. The microfluidic device of claim 118, further comprising:
<Claim 120>
Microchannel containing control reagents
120. The microfluidic device of claim 118 or 119, further comprising:
<Claim 121>
The control reagent includes the detectable label and the capture agent.
121. The microfluidic device of claim 120.
<Claim 122>
the amino acid sequence of the RBD is at least 75%, 80%, 85%, 90%, 95%, 96% of amino acids 319-541 of the SARS-CoV-2 spike protein (SEQ ID NO: 1); have 97%, 98%, 99%, 99.5, or 100% sequence identity
122. The microfluidic device according to any one of claims 118 to 121.
<Claim 123>
The RBD or S1 subunit of the coronavirus spike protein, or a fragment thereof further comprises an Fc region.
123. A microfluidic device according to any one of claims 118 to 122.
<Claim 124>
A method for detecting coronavirus antigens in a sample from a subject, the method comprising:
applying said sample to a binding assay comprising a first reagent and a second reagent;
Equipped with
The first reagent includes an antibody against a coronavirus antigen,
the first reagent is labeled with a detectable label or capture agent;
the second reagent is attached to a detectable label or capture agent;
The first reagent and the second reagent are capable of binding to the coronavirus antigen and forming a complex comprising the first reagent, the coronavirus or coronavirus antigen, and the second reagent.
A method characterized by:
<Claim 125>
The second reagent includes a second antibody against the coronavirus antigen.
125. The method of claim 124.
<Claim 126>
The antigen is the spike protein, nucleocapsid protein, envelope protein, membrane protein, or hemagglutinin-esterase dimeric protein of the coronavirus.
126. The method according to claim 124 or 125.
<Claim 127>
the antigen is nucleocapsid protein
127. The method of claim 126.
<Claim 128>
The first reagent is bound or configured to bind a detectable label.
128. A method according to any one of claims 124 to 127.
<Claim 129>
The first reagent is bound to or configured to bind to a capture agent.
128. A method according to any one of claims 124 to 127.
<Claim 130>
The second reagent is bound or configured to bind a detectable label.
130. The method according to any one of claims 124 to 127, 129.
<Claim 131>
The second reagent is bound or configured to bind to a capture agent.
131. A method according to any one of claims 124 to 128, 130.
<Claim 132>
The detectable label includes a fluorescent label, such as fluorescent latex beads.
132. A method according to any one of claims 124 to 131, characterized in that:
<Claim 133>
The capture agent includes magnetic beads.
133. A method according to any of claims 124 to 132.
<Claim 134>
The method is carried out in a microfluidic device according to any of claims 72 to 89.
134. A method according to any one of claims 124 to 133.
<Claim 135>
The coronavirus is SARS-CoV-2
135. A method according to any one of claims 124 to 134.
<Claim 136>
The samples include blood, serum, plasma, saliva, mucus, and/or specimens taken from throat, nasopharyngeal, or nasal swabs.
136. A method according to any of claims 124 to 135.
<Claim 137>
Before applying the sample to the binding assay, the sample is contacted with latex particles.
137. A method according to any one of claims 124 to 136.
<Claim 138>
Before applying the sample to the binding assay, the sample is contacted with a buffer containing a salt solution.
138. A method according to any one of claims 124 to 137.
<Claim 139>
When applying said sample to said binding assay, the presence of said coronavirus antigen is detected.
139. A method according to any one of claims 124 to 138.
<Claim 140>
A microfluidic device for detecting coronavirus antigens in a sample from a subject, the device comprising:
a microchannel containing a dried first reagent and a second reagent therein;
Equipped with
The first reagent includes an antibody against a coronavirus antigen,
the first reagent is bound or configured to bind a detectable label or capture agent;
the second reagent is bound or configured to bind a detectable label or capture agent;
The first reagent and the second reagent, when solubilized by the sample, form a complex comprising the first reagent, the coronavirus antigen, and the second reagent.
A microfluidic device characterized by:
<Claim 141>
The second reagent includes an antibody against the coronavirus antigen.
141. The microfluidic device of claim 140.
<Claim 142>
The antigen includes the spike protein, nucleocapsid protein, envelope protein, membrane protein, or hemagglutinin-esterase dimeric protein of the coronavirus.
142. The microfluidic device according to claim 140 or 141.
<Claim 143>
The first reagent is bound or configured to bind a detectable label.
143. A microfluidic device according to any one of claims 140 to 142.
<Claim 144>
The first reagent is bound to or configured to bind to a capture agent.
143. A microfluidic device according to any one of claims 140 to 142.
<Claim 145>
The second reagent is bound or configured to bind a detectable label.
145. The microfluidic device according to any one of claims 140 to 142 and 144.
<Claim 146>
The second reagent is bound or configured to bind to a capture agent.
144. The microfluidic device according to any one of claims 140 to 143.
<Claim 147>
The detectable label includes a fluorescent label, such as fluorescent latex beads.
147. A microfluidic device according to any one of claims 140 to 146.
<Claim 148>
The capture agent includes magnetic beads.
148. A microfluidic device according to any one of claims 140 to 147.
<Claim 149>
The coronavirus is SARS-CoV-2
149. A microfluidic device according to any one of claims 140 to 148.
<Claim 150>
The samples include blood, serum, plasma, saliva, mucus, and/or specimens taken from throat, nasopharyngeal, or nasal swabs.
150. The microfluidic device according to any one of claims 140 to 149.
<Claim 151>
a second microfluidic channel containing the same reagents as the first and second reagents;
151. The microfluidic device according to any one of claims 140 to 150, further comprising:
<Claim 152>
a second or third microfluidic channel containing reagents for binding antibodies to coronavirus antigens;
152. The microfluidic device according to any one of claims 140 to 151, further comprising:
<Claim 153>
The reagent for binding the antibody to the coronavirus antigen includes:
a first reagent comprising a receptor binding domain (RBD) of coronavirus spike protein, or a fragment thereof;
a second reagent;
including;
the first reagent is bound or configured to bind a detectable label or capture agent;
the second reagent comprises an anti-immunoglobulin antibody bound or configured to bind a detectable label or capture agent;
the first reagent and the second reagent bind to the anti-coronavirus spike protein antibody to form a complex comprising the first reagent, the anti-coronavirus spike protein antibody, and the second reagent;
Formation of said complex indicates the presence of said antibody against said coronavirus antigen within said sample.
153. The microfluidic device of claim 152.
<Claim 154>
The anti-immunoglobulin antibody is an anti-IgA antibody or an anti-IgG antibody.
154. The microfluidic device of claim 153.
<Claim 155>
The anti-immunoglobulin antibody is an anti-IgA antibody.
155. The microfluidic device of claim 154.
<Claim 156>
2nd, 3rd or 4th microchannel containing control reagents
156. The microfluidic device according to any one of claims 145 to 155, further comprising:
<Claim 157>
(a) a microfluidic device defining a microfluidic channel network therein;
(b) a supply electrode including a supply contact, a supply lead, and a supply portion;
(c) a sensing electrode including a sensing contact, a sensing lead including a plurality of sensing lead portions, and a plurality of liquid sensing portions;
Equipped with
each of the supply contacts and the supply leads are located outside of the microfluidic channel network;
the supply lead extends along the microfluidic device from the supply contact to the supply portion disposed at a supply location within the microfluidic channel network;
(i) the sensing contacts and each sensing lead portion are located outside the microfluidic channel network;
(ii) each liquid sensing portion is disposed within the microfluidic channel network at a respective liquid sensing location;
Each liquid sensing location is (a) spaced apart from the supply location and other fluid sensing locations, and (b) electrically connected to the other fluid sensing locations via at least one of the sensing lead portions. is in a conductive state
A product characterized by:
<Claim 158>
the microfluidic channel network includes a plurality of channels;
each of the at least plurality of liquid sensing locations being disposed within a different channel of the plurality of channels;
158. The product of claim 157.
<Claim 159>
the sensing electrodes include N consecutive sensing pairs;
each sensing pair includes at least one of the sensing lead portions and at least one of the liquid sensing portions disposed within a respective one of the plurality of channels;
The N is at least 2, at least 3, at least 4, or at least 5.
159. The product of claim 158.
<Claim 160>
The liquid sensing portion of each of the N consecutive sensing pairs is disposed within a different respective one of the plurality of N channels.
160. The product of claim 159.
<Claim 161>
the sensing lead includes first and second sensing lead branches;
Each of the first and second sensing lead branches includes at least one of the liquid sensing portions disposed within a respective different channel of the microfluidic channel network.
161. A product according to any one of claims 157 to 160.
<Claim 162>
The first sensing lead branch includes a plurality of the sensing lead portions.
162. The product of claim 161.
<Claim 163>
The microfluidic channel network includes an electrically conductive liquid that establishes electrical communication between the supply portion and at least one, such as all, of the plurality of liquid sensing portions.
161. A product according to any one of claims 157 to 160.
<Claim 164>
The conductive liquid is selected from the group consisting of blood-based liquids, whole blood, fingerstick blood, venous blood, plasma, nasopharyngeal samples, saliva, sputum, urine, buffers, or combinations thereof. Contains samples
164. The product of claim 163.
<Claim 165>
The total volume of the conductive liquid within the microfluidic channel network is about 100 μL or less, about 50 μL or less, about 25 μL or less, about 15 μL or less, or about 10 μL.
165. A product according to claim 163 or 164.
<Claim 166>
with any of the readers disclosed herein;
166. The article of any of claims 157-165 received within the reader;
A system characterized by:
<Claim 167>
(a) inputting an electrical supply signal to a conductive liquid present at the supply location within the microfluidic channel network at a supply location within the microfluidic channel network;
(b) determining an electrical output signal at the sensing contact of the sensing electrode;
Equipped with
The sensing electrode is
(i) a conductive sensing lead;
(ii) defining a first liquid sensing location within the microfluidic channel network in electrical communication with the sensing contact via the sensing lead; a first liquid sensing portion configured to be in electrical communication with the conductive liquid when present in the conductive liquid;
(ii) in electrical communication with the sensing contact to define a second liquid sensing location within the microfluidic channel network, such that the electrical conductivity when present within the microfluidic channel network at the second liquid sensing location; a second liquid sensing portion configured to be in electrical continuity with the sexual liquid;
including;
(a) Each of the supply position, the first liquid sensing position, and the second liquid sensing position is spaced apart from the other supply position, the first liquid sensing position, and the second liquid sensing position. Ori,
(b) the supply location and the sensing electrode are arranged in the microfluidic channel network in the absence of a conductive liquid extending from the supply location to at least one of the first and second liquid sensing locations; are electrically isolated from each other,
The method further includes:
(c) based on the determination of the electrical output signal, the conductive liquid is present at the dispensing location and extends therefrom within the microfluidic channel network to at least one of the first and second liquid sensing locations; The process of determining whether or not
A method characterized by comprising:
<Claim 168>
The microfluidic channel network is located within a microfluidic device.
168. The method of claim 167.
<Claim 169>
The microfluidic device includes a supply electrode;
A feed portion of the feed electrode is disposed within the microfluidic channel network to define the feed location.
169. The method of claim 168.
<Claim 170>
the supply electrode includes a supply contact and a supply lead;
the supply contact and the supply lead are each located outside the microfluidic channel network;
the supply contact is in electrical communication with the supply portion via the supply lead;
The inputting step includes inputting the electrical supply signal to the supply contact.
170. The method of claim 169.
<Claim 171>
(i) the microchannel is a first analysis channel;
(ii) the microfluidic device includes a sample application port and a supply channel disposed between and in fluid communication with the sample application port and the first analysis channel;
157. A method according to any one of claims 140 to 156.
<Claim 172>
The microfluidic device includes at least one zone of dry anticoagulant disposed within the sample application port, the supply channel, or a combination thereof.
172. The microfluidic device of claim 171.
<Claim 173>
At least one zone of soluble dry anticoagulant comprises:
(i) within or adjacent to the sample application port, or both;
(ii) a length within said feed channel that is essentially free or free of soluble dry anticoagulant, such as at least about 3 mm, at least about 5 mm, at least about 7 mm; spaced from the sample application port by a length of 5 mm, or at least about 10 mm;
is placed
173. The microfluidic device of claim 172.
<Claim 174>
at least one zone of dry anticoagulant is located within or adjacent to the sample application port;
The microfluidic device is disposed within the supply channel for a length of the supply channel that is essentially free or free of soluble dry anticoagulant, e.g., at least about 3 mm, at least about 5 mm, a second zone of soluble dry anticoagulant spaced apart from the first zone of dry anticoagulant by a length of at least about 7.5 mm, or at least about 10 mm;
174. The microfluidic device of claim 173.
<Claim 175>
The dry anticoagulant comprises lithium heparin or consists essentially of lithium heparin.
175. The microfluidic device according to any one of claims 172 to 174.
<Claim 176>
heating the sample to between about 37°C and about 47°C during at least a portion of applying the sample to a binding assay;
140. The method of any of claims 124-139, further comprising:
<Claim 177>
heating the sample to between about 40° C. and about 44° C. during at least a portion of applying the sample to a binding assay;
177. The method of claim 176, further comprising:
<Claim 178>
heating the sample to about 42° C. during at least a portion of applying the sample to a binding assay;
178. The method of claim 177, further comprising:
<Claim 179>
At least substantially all, essentially all, or all of the steps of applying the sample to a binding assay are performed within a microfluidic channel network of a microfluidic device.
179. A method according to any one of claims 124-139, 176-178.
<Claim 180>
The method is
introducing the sample into a sample port of the microfluidic channel network;
further comprising;
The step of applying the sample to a binding assay includes flowing at least a first portion of the sample along a supply channel in fluid communication with the sample port.
180. The method of claim 179.
<Claim 181>
The introducing step and/or the flowing step includes contacting the first portion of the sample with a soluble dry anticoagulant disposed within the sample port and/or the supply channel.
including
181. The method of claim 180.
<Claim 182>
The contacting step includes:
contacting the first portion of the sample with a soluble dry anticoagulant disposed within and/or adjacent the supply channel;
flowing the sample along the length of the feed channel essentially free or free of dry anticoagulant;
thereafter contacting the first portion of the sample with a second amount of soluble dry anticoagulant disposed within the supply channel;
including
181. The method of claim 180.
<Claim 183>
flowing the first portion of the sample along the length of the feed channel essentially free or free of soluble dry anticoagulant;
The leading edge of the first portion of the sample is at least about 3 mm along the length of the feed channel before the leading edge contacts the second amount of soluble dry anticoagulant. 5 mm, at least about 7.5 mm, or at least about 10 mm.
183. The method of claim 182.
<Claim 184>
The soluble dry anticoagulant comprises or consists essentially of lithium heparin.
184. The microfluidic device according to any one of claims 181 to 183.
<Claim 185>
The method provides for producing influenza antigens within about 15 minutes, within about 12.5 minutes, within about 11.5 minutes, or within about 10.5 minutes of flowing the sample along the supply channel. , a coronavirus antigen, such as a SARS-CoV-2 antigen, and combinations thereof.
185. A method according to any of claims 180 to 184.
<Claim 186>
The step of applying the sample to the binding assay includes mixing a portion of the sample with the first and second reagents;
The total volume of the sample mixed with the first and second reagents is about 5 μL or less, about 4 μL or less, about 3 μL or less, about 2.5 μL or less, about 2 μL or less, or about 1.75 μL or less.
186. A method according to any of claims 180 to 185.
<Claim 187>
The total volume of the sample mixed with the first and second reagents comprises at least a portion of the sample contacted with a soluble dry anticoagulant.
187. The method of claim 186.
<Claim 188>
The step of applying the sample to the binding assay comprises:
contacting the sample with at least one of the first and second reagents within the microfluidic channel network of the microfluidic device;
oscillating the gas pressure at the liquid-gas interface of the sample at at least one frequency for an oscillation duration when the sample is in contact with at least one of the first and second reagents;
including
188. A method according to any of claims 179 to 187.
<Claim 189>
The at least one frequency is an acoustic frequency, such as a frequency between about 900 and 1300 Hz, between about 1000 and 1200 Hz, or between about 1050 and 1150 Hz.
189. The method of claim 188.
<Claim 190>
The vibration duration is between about 5 and 60 seconds, between about 10 and 50 seconds, between about 15 and 40 seconds, or between about 20 and 30 seconds.
200. A method according to claim 188 or 189, characterized in that.
<Claim 191>
The step of oscillating the pressure of the gas at the at least one frequency is between about 1% and 25%, between about 2.5% and 15%, or between about 2.5% and 15% of the average frequency of the oscillations during the oscillation duration. , between about 5 and 12.5%, periodically varying said at least one frequency, such as by a triangular wave, a square wave or a sine wave, over a frequency range of between about 5 and 12.5%.
191. A method according to any one of claims 188 to 190.
<Claim 192>
The step of varying is performed periodically,
The period of the cyclically varying step is between about 1% and about 25%, between about 2% and about 20%, or between about 3% and about 15% of the vibration duration. be
192. A method according to any of claims 188 to 191, characterized in that:
<Claim 193>
The vibration duration is about 25 seconds,
the average frequency of vibration during the vibration duration is about 1100 Hz;
The frequency range of vibration during the vibration duration is about 100 Hz (about 1050 Hz to about 1050 Hz),
The periodic variation step is performed as a sine wave or a triangular wave with a period of about 1.5 seconds.
194. A method according to any one of claims 191 to 193.
<Claim 194>
The step of changing
(a) is executed periodically, or
(b) implemented as a linear or non-linear ramp increasing or decreasing during said vibration duration;
Said step of cyclically varying is performed N times during said vibration duration, N=x×t_osc/t_perand
x is at least about 0.05, at least about 0.1, at least about 0.25, at least about 0.5, at least about 0.75, at least about 0.9, at least about 0.95, or at least about 0 .975, and
t_oscis the vibration duration,
t_peris the period of the process that is changed periodically
194. A method according to any one of claims 191 to 193.
<Claim 195>
the gas at the liquid-gas interface is encapsulated within a chamber of the microfluidic device;
The step of oscillating the pressure of the gas is carried out by oscillating the position of a portion of the wall of the chamber at the at least one frequency.
195. A method according to any of claims 188 to 194.
<Claim 196>
The step of vibrating the position of the portion of the wall includes the step of vibrating an internal dimension of the chamber, such as a height or a width, at the at least one frequency.
196. The method of claim 195.
<Claim 197>
The step of vibrating the position of the portion of the wall includes vibrating the internal dimensions of the wall by at least about ±5 μm, at least about ±7.5 μm, or at least about ±10 μm.
197. A method according to claim 195 or 196.
<Claim 198>
The step of vibrating the position of the portion of the wall includes vibrating the internal dimensions of the wall by about ±35 μm or less, about ±30 μm or less, or about ±25 μm.
197. A method according to claim 195 or 196.
<Claim 199>
The step of vibrating the position of the portion of the wall includes the step of vibrating the volume of gas at the liquid-gas interface at the at least one frequency.
200. A method according to any of claims 195 to 198.
<Claim 200>
The step of vibrating the volume of the gas comprises at least about ±5%, at least about ±7.5%, at least about ±10%, at least about ±15%, of the average total volume of the gas during the vibration cycle; or vibrating said volume by at least about ±20%.
200. The method of claim 199.
<Claim 201>
The step of vibrating the volume of the gas can be performed by no more than about ±75%, no more than about 50%, no more than about 35%, or no more than about 27.5% of the average total volume of the gas during the vibrating cycle. including the step of vibrating the volume.
200. The method of claim 199.
<Claim 202>
The step of oscillating the pressure of the gas at the liquid-gas interface comprises a total relative amount of at least about 5%, at least about 10%, at least about 20%, at least about 25%, or at least about 35%. (((P_max-P_min)/P_avg)×100), oscillating the pressure of the gas peak-to-peak by
P_maxis the maximum gas pressure during the oscillation cycle,
P_minis the minimum gas pressure during the oscillation cycle,
P_avgis the average gas pressure during the vibration cycle
202. A method according to any one of claims 188 to 201, characterized in that.
<Claim 203>
The step of oscillating the pressure of the gas at the liquid-gas interface comprises a total relative amount (((P_max-P_min)/P_avg)×100) to oscillate the pressure of the gas peak-to-peak.
203. A method according to any of claims 188 to 202.
<Claim 204>
The step of oscillating the pressure of the gas at the liquid-gas interface comprises a total amount (P_max-P_min) oscillating the pressure of the gas peak-to-peak by
204. A method according to any one of claims 188 to 203.
<Claim 205>
The step of oscillating the pressure of the gas at the liquid-gas interface includes a total amount (P_max-P_min) oscillating the pressure of the gas peak-to-peak by
205. A method according to any of claims 188 to 204.
<Claim 206>
The step of applying the sample to the binding assay comprises:
(i) contacting the sample with at least one of the first and second reagents within the microfluidic channel network of the microfluidic device;
(ii) moving the liquid-gas interface of the sample in a first direction along the channels of the microfluidic channel network;
(iii) sensing when the liquid-gas interface of the sample contacts an electrode disposed within the channel;
(iv) stopping movement of the sample in the first direction along the channel;
206. A method according to any one of claims 179 to 205, comprising:
<Claim 207>
The electrode is a first electrode,
The method includes, after the step of stopping movement in the first direction,
(i) along said channel in a second direction opposite to said first direction until said liquid-gas interface passes beyond the location of a second electrode disposed within said channel; moving the liquid-gas interface of the sample;
(ii) sensing, via the second electrode, that the liquid-gas interface has passed beyond the second electrode;
(iii) stopping movement of the sample in the second direction along the channel;
207. The method of claim 206, further comprising:
<Claim 208>
(a) repeating steps (ii) to (iv) according to claim 206;
(b) thereafter repeating steps (i) to (iii) according to claim 207;
208. The method of claims 206 and 207, further comprising:
<Claim 209>
The step of moving the sample in the first direction includes increasing the volume occupied by the gas at the liquid-gas interface,
Moving the sample in the second opposite direction includes reducing the volume occupied by the gas.
209. A method according to any one of claims 206 to 208, characterized in that:
<Claim 210>
The total time for (a) performing steps (ii) to (iv) of claim 206 and (b) subsequently performing steps (i) to (iii) of claim 207 is about 2 to for 8 seconds, for about 3 to 7 seconds, for about 4 to 6 seconds, or for about 4.5 to 5.5 seconds.
210. A method according to any one of claims 206 to 209, characterized in that:
<Claim 211>
In carrying out steps (ii)-(iv) of claim 191, the total volume of gas in the channel displaced by the liquid at the liquid-gas interface is between about 75 and 1000 nL. between about 150 and 750 nL, between about 250 and 550 nL, or between about 300 and 500 nL
211. A method according to any one of claims 206 to 210, characterized in that:
<Claim 212>
In performing steps (ii) to (iv) of claim 191, the total distance along the channel traversed by the liquid-gas interface is between about 2 and 10 mm, between about 3 and 9 mm. between about 4 to 8 mm, between about 4 to 7 mm, or between about 4 to 6 mm
211. A method according to any one of claims 206 to 210, characterized in that:
<Claim 213>
The first and second electrodes are separated by a distance of between about 2 and 10 mm, between about 3 and 9 mm, between about 4 and 8 mm, between about 4 and 7 mm, or between about 4 and 6 mm. spaced apart along the longitudinal axis of the channel
213. A method according to any one of claims 207 to 212, characterized in that:
<Claim 214>
The total volume of the gas at the liquid-gas interface is between about 1 μL and about 25 μL, between about 2.5 μL and about 20 μL, between about 3.5 μL and about 15 μL, between about 3.5 μL and about 10 μL. or between about 3.5 μL and about μL.
214. A method according to any one of claims 188 to 213, characterized in that:
<Claim 215>
The sample includes blood, serum, or plasma,
For example, the sample comprises or consists essentially of serum and/or plasma.
140. A method according to any of claims 124 to 139.
<Claim 216>
The step of applying the sample to a binding assay may lyse, for example, white blood cells, red blood cells, or viruses, such as coronaviruses such as SARS-CoV-2, in the sample without subjecting the sample to lysis. carried out without subjecting said sample to a sufficient lysis step to cause
216. The method of claim 215.
<Claim 217>
The step of applying the sample to a binding assay may be performed without releasing coronavirus antigens from the cells present in the sample, for example from within white blood cells, from within red blood cells, or from either white blood cells or red blood cells. Performed without releasing coronavirus antigens
216. The method of claim 215.
<Claim 218>
The step of applying the sample to a binding assay may include, for example, first applying the sample to the walls of cells present within the sample, such as white blood cells, red blood cells, etc., without first contacting the sample with a chemical lysis reagent. or without contact with alkali, detergents, or enzymes of sufficient concentration to rupture the walls of either white blood cells or red blood cells.
216. The method of claim 215.
<Claim 219>
The step of applying the sample to a binding assay may include, for example, first dissolving the sample into cells present within the sample, such as white blood cells, red blood cells, red blood cells, etc., without first subjecting the sample to a physical lysis step. or without exposure to thermal conditions, osmotic pressure, shear forces or cavitation sufficient to rupture the walls of either white blood cells or red blood cells.
216. The method of claim 215.
<Claim 220>
The step of applying the sample to a binding assay may be performed without first subjecting the sample to a lysis step sufficient to lyse, e.g. deenvelop or inactivate, the coronavirus within the sample, e.g. , without first subjecting said sample to a lysis step sufficient to dissolve the SARS-CoV-2 present within said sample.
216. The method of claim 215.
<Claim 221>
upon applying said sample to a binding assay, the presence of said coronavirus antigen is detected;
Substantially all of the detected coronavirus antigens are free antigens, eg, antigens not associated with the complete virus.
221. The method of any of claims 215-220.
<Claim 222>
The sample comprises or consists essentially of serum and/or plasma.
222. The method of any of claims 215-221.
<Claim 223>
The method includes agglutinating red blood cells in a volume of blood to prepare the sample.
223. The method of claim 222.
<Claim 224>
The step of agglutinating the red blood cells includes contacting the volume of blood with antibodies directed against proteins produced by or otherwise associated with red blood cells, such as antibodies against glycophorin A.
224. The method of claim 223.
<Claim 225>
The step of agglutinating red blood cells includes contacting the volume of blood with an agglutinating protein, such as phytohemagglutinin E.
225. A method according to claim 223 or 224, characterized in that.
<Claim 226>
All or substantially all of the steps of applying the sample to the binding assay are performed within a microfluidic device.
226. The method of any of claims 222-225.
<Claim 227>
Said step of aggregating is performed within a microfluidic device
227. The method of any of claims 223-226.
<Claim 228>
The method is
introducing the volume of blood into the microfluidic device;
contacting the blood with the antibody of claim 224 or the aggregating protein of claim 225 within a channel of the microfluidic device;
including
228. The method of claim 227.
<Claim 229>
The method is
Separation of plasma and/or serum samples from red blood cells
including
229. A method according to claim 227 or 228.
<Claim 230>
Said step of separating the plasma and/or serum sample is carried out without passing the plasma and/or serum through a filter.
230. The method of claim 229.
<Claim 231>
Said step of separating plasma and/or serum samples is performed in a microfluidic channel having a substantially smooth internal surface.
231. The method of claim 229 or 230.
<Claim 232>
Said step of separating the plasma and/or serum sample comprises:
A microfluidic channel having an interior surface free of protrusions having a height greater than about 10%, 7.5%, 5%, or about 2.5% relative to the width or height of the microfluidic channel. executed within a portion of
232. A method according to any of claims 229 to 231, characterized in that:
<Claim 233>
Said step of separating a sample of plasma and/or serum is such that the movement of red blood cells along the longitudinal axis of said microfluidic channel is retarded relative to the movement of plasma and/or serum along said longitudinal axis. carried out within a portion of a microfluidic channel that has an internal surface free of structured protrusions.
233. The method of any of claims 229-232.
<Claim 234>
Said step of separating plasma and/or serum samples is performed within a portion of a microfluidic channel having at least one internal turn of at least about 90 degrees.
235. The method of any of claims 229-234.
<Claim 235>
The microfluidic device is a microfluidic device according to any one of claims 140 to 156 and claims 171 to 175.
235. The method of any of claims 226-234.
<Claim 236>
The sample is a sample obtained from a human infected with SARS-CoV-2 or a human suspected of being infected with SARS-CoV-2.
237. The method of any of claims 215-236.
<Claim 237>
The human is asymptomatic
237. The method of claim 236.
<Claim 238>
The person does not exhibit difficulty breathing or bluish lips or face.
237. The method of claim 236.
<Claim 239>
The sample obtained from the human is obtained within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, or within 2 days from the onset of symptoms.
239. The method of claim 236 or 238.
<Claim 240>
The sample obtained from said person is obtained by the date of onset of symptoms.
240. The method of claim 236, 238 or 239, characterized in that.
<Claim 241>
The sample obtained from the human was obtained before the occurrence of seroconversion regarding SARS-CoV-2.
241. The method of any of claims 236-240.
<Claim 242>
The antigen is the spike protein, nucleocapsid protein, envelope protein, membrane protein, or hemagglutinin-esterase dimeric protein of SARS-CoV-2.
242. The method of any of claims 215-241.
<Claim 243>
(a) mixing a blood sample containing red blood cells with an agglutination reagent;
(b) in a microchannel of a microfluidic device, the mixed blood sample and the agglutination reagent are placed in a red blood cell portion disposed in a first portion of the microchannel and in a second portion of the microchannel; a step of separating into a plasma portion;
Equipped with
the red blood cell portion comprises essentially all of the red blood cells of the blood sample;
Said plasma portion consists essentially of plasma of said blood sample.
A method characterized by:
<Claim 244>
The blood sample is a whole blood sample of a mammal, e.g. a human.
244. The method of claim 243.
<Claim 245>
The step of mixing is performed within the microchannel of the microfluidic device.
245. A method according to claim 243 or 244, characterized in that.
<Claim 246>
(i) the microfluidic device includes a liquid sample introduction port in fluid communication with the microchannel, the microchannel including the agglutination reagent disposed therein;
(ii) the step of mixing includes introducing the blood sample into the microchannel via the liquid sample introduction port, flowing the blood sample along the microchannel, and mixing the agglutination reagent and the blood sample.
246. The method of claim 245.
<Claim 247>
The separating step includes sequentially forming the red blood cell portion and the plasma portion along the microchannel.
247. The method of any of claims 243-246.
<Claim 248>
The method includes forming a distal liquid-gas interface disposed within the microchannel and spaced apart from an ambient gas surrounding the microfluidic device by at least the red blood cell portion and the plasma portion.
including;
The liquid at the distal liquid-gas interface is one of the red blood cell portion or the plasma portion.
248. The method of claim 247.
<Claim 249>
The liquid at the distal liquid-gas interface is plasma of the plasma portion.
249. The method of claim 248.
<Claim 250>
the step of separating includes forming a liquid-liquid interface between the red blood cell portion and the plasma portion;
One of the liquids at the liquid-liquid interface is the liquid of the red blood cell portion,
The other liquid at the liquid-liquid interface is the liquid of the plasma portion.
250. A method according to any of claims 247 to 249.
<Claim 251>
mixing the plasma of the plasma portion with one or more reagents disposed within the microchannel;
Equipped with
The one or more reagents are configured to interact with a target present in the plasma portion.
251. The method of claim 250.
<Claim 252>
The one or more reagents include at least one reagent configured to participate in a binding reaction with the target, such as an immunological reaction with the target, such as an antibody configured to bind to the target. or a fragment thereof, containing
252. The method of claim 251.
<Claim 253>
determining the presence and/or amount of the target within the plasma portion based at least in part on the interaction of the at least one reagent with the target;
253. The method of claim 251 or 252, further comprising:
<Claim 254>
maintaining the liquid-liquid interface during mixing of the plasma of the plasma portion with the one or more reagents disposed within the microchannel;
254. A method according to any one of claims 251 to 253, comprising:
<Claim 255>
maintaining the liquid-liquid interface during the step of determining the presence and/or amount of the target within the plasma portion;
255. The method of claim 254, comprising:
<Claim 256>
The step of separating the mixed blood sample and the agglutination reagent comprises:
flowing the mixed blood sample and the agglutination reagent in a first direction along the microchannel;
thereafter flowing the mixture in a second direction opposite to the first direction along the microchannel;
including
256. The method of any of claims 243-255.
<Claim 257>
The step of separating the mixed blood sample and the agglutination reagent comprises:
repeating the step of flowing the mixed blood sample and the agglutination reagent in the first direction, and then the step of flowing the mixture in the second direction at least N times;
including;
The N is at least about 3, at least about 5, at least about 7, or at least about 10.
250. The method of claim 249.
<Claim 258>
The N is about 20 or less, about 15 or less, or about 10 or less.
251. The method of claim 250.
<Claim 259>
Said step of separating is carried out without passing said plasma portion through a filter, e.g. a membrane.
259. The method of any of claims 243-258.
<Claim 260>
Said step of separating may include, for example, subjecting said blood sample to a deterministic lateral displacement sufficient to separate said blood sample into said red blood cell portion and said plasma portion. carried out without exposure to
260. The method of any of claims 243-259.
<Claim 261>
The internal surface of the portion of the microchannel in which the step of separating is carried out has projections or microstructures sufficient to preferentially retain red blood cells in sufficient quantity to separate the red blood cell portion and the plasma portion. substantially free of
261. The method of any of claims 243-260.
<Claim 262>
Said step of separating may be performed without subjecting said blood sample to sufficient inertial focusing to separate said red blood cell portion and said plasma portion, e.g., without subjecting said blood sample to essentially any inertial focusing. is executed with
262. The method of any of claims 243-261.
<Claim 263>
Said step of separating may be performed without subjecting said blood sample to centrifugal force sufficient to separate said red blood cell portion and said plasma portion, e.g., without subjecting said blood sample to essentially any centrifugal force. is executed with
263. The method of any of claims 243-262.
<Claim 264>
The step of separating is performed without rotating the microfluidic device.
264. The method of any of claims 243-263.
<Claim 265>
The step of separating is performed without flowing the blood sample along a curved flow path within the microchannel.
265. The method of any of claims 243-264.
<Claim 266>
Said step of separating is performed with the flow axis of said microchannel oriented substantially perpendicular to the Earth's local gravitational field, e.g., at about 20 degrees to the Earth's local gravitational field. with the flow axis of the microchannel oriented vertically or essentially vertically within about 15 degrees, within about 10 degrees, or within about 5 degrees.
266. The method of any of claims 243-265.
<Claim 267>
The agglutination reagent is
proteins that induce or promote aggregation, such as phytohemagglutinin,
antibodies that induce or promote aggregation, such as anti-glycophorin A antibodies, and
Lectins, such as lectins from legumes, such as soy agglutinin from soybeans,
including one or more of
267. The method of any of claims 243-266.
<Claim 268>
The volume of the plasma portion separated from the blood sample is at least about 0.075 μL, at least about 0.1 μL, at least about 0.15 μL, at least about 0.175 μL, or at least about 0.2 μL.
268. The method of any of claims 243-267.
<Claim 269>
The volume of the plasma portion separated from the blood sample is about 0.75 μL or less, about 0.65 μL or less, about 0.55 μL or less, about 0.45 μL or less, about 0.4 μL or less, about 0.35 μL or less, or approximately 0.325 μL or less
269. The method of any of claims 243-268.
<Claim 270>
(a) introducing a blood sample, e.g. a whole blood sample from a mammal such as a human, into a microchannel of the microfluidic device;
(b) mixing the blood sample with an agglutination reagent within the microchannel;
(c) disposing the mixed blood sample and the agglutination reagent within the microchannel of the microfluidic device with a red blood cell portion disposed in a first portion of the microchannel and a second portion of the microchannel; a step of separating said red blood cell portion and said plasma portion into contact at an interface therebetween;
(d) mixing the plasma of the plasma portion within the microchannel of the microfluidic device with a reagent, such as an immunological reagent, configured to bind to a target present within the plasma;
(e) determining the presence and/or amount of the target within the plasma of the plasma portion while maintaining the contact between the red blood cell portion and the plasma portion at the interface;
Equipped with
the red blood cell portion comprises essentially all of the red blood cells of the blood sample;
Said plasma portion consists essentially of plasma of said blood sample.
A method characterized by:
<Claim 271>
(a) separating a blood sample, e.g. a whole blood sample, into a red blood cell portion and a plasma portion, the red blood cell portion and the plasma portion being connected by a liquid interface therebetween;
(d) mixing the plasma of the plasma portion with a reagent, such as an immunological reagent, configured to facilitate the determination of targets present within the plasma;
(e) determining the presence and/or amount of the target within the plasma of the plasma portion while maintaining contact between the red blood cell portion and the plasma portion at the interface;
Equipped with
the red blood cell portion comprises essentially all of the red blood cells of the blood sample;
Said plasma portion consists essentially of plasma of said blood sample.
A method characterized by:

Sequence list
Array ID NO: 1

Claims (24)

A method for detecting at least one target substance in a sample liquid, the method comprising:
(a) introducing a sample liquid into a microfluidic channel of a microfluidic device, the microfluidic channel containing a gas therein, and the sample liquid contacting the gas, thereby causing their a step of forming a sample liquid-gas interface between the two;
(b) moving the sample liquid and the sample liquid-gas interface to a first zone by reducing the pressure of the gas, the first zone being a first zone disposed therein; A process of accommodating reagents,
(c) mixing the sample liquid with the first reagent to form a first mixture by oscillating the pressure of the gas;
Equipped with
The step of oscillating the pressure of the gas is different from the step of reducing the pressure of the gas,
i) is carried out before the latter;
ii) be carried out simultaneously with the latter;
iii) carried out after the latter; or
iv) A method characterized in that it is carried out before, simultaneously with, and after the latter. The step of vibrating the pressure of the gas is performed at a frequency of 2000 Hz or less, a frequency of 1500 Hz or less, a frequency of 1250 Hz or less, a frequency of 1000 Hz or less, a frequency of 900 Hz or less, a frequency of 800 Hz or less, a frequency of 5 to 2500 Hz, or a frequency of 5 to 2500 Hz. The method according to claim 1, characterized in that it is carried out at a frequency of ~2000 Hz. 2. The method of claim 1, wherein the first reagent comprises a lysis reagent, a binding reagent, or an optical label. Reducing the pressure of the gas includes increasing an internal spacing between a first inner wall and a second inner wall of a bladder region located at a distal portion of the microfluidic device;
The bladder region, the first inner wall, and the second inner wall are in direct contact with the gas and in gas communication with the sample liquid-gas interface and the microfluidic channel. The method according to claim 1. 5. The step of oscillating the pressure of the gas includes the step of oscillating the internal spacing between the first inner wall and the second inner wall at one or more acoustic frequencies. The method described in 4. oscillating the internal spacing includes varying the internal spacing over a total peak-to-peak distance of 5 μm to 75 μm;
6. The method of claim 5, wherein the total peak-to-peak distance is measured along an axis perpendicular to a plane defined by the microfluidic device. (a) moving the first mixture and the sample liquid-gas interface from the first zone to a second zone by reducing the pressure of the gas again, the second zone comprising: accommodating a second reagent disposed therein;
(b) mixing the first mixture with the second reagent to form a second mixture by again oscillating the pressure of the gas;
further comprising;
The step of oscillating the pressure of the gas again is the step of reducing the pressure of the gas again,
i) is carried out before the latter;
ii) carried out simultaneously with the latter;
iii) carried out after the latter; or
6. A method according to claim 5, characterized in that iv) is carried out before, simultaneously with and after the latter. (a) moving the second mixture and the sample liquid-gas interface from the second zone to a third zone by further reducing the pressure of the gas again, the third zone being , containing a third reagent disposed therein;
(b) mixing the second mixture with the third reagent to form a third mixture by further oscillating the pressure of the gas;
further comprising;
The step of further oscillating the pressure of the gas again is the step of further reducing the pressure of the gas again,
i) is carried out before the latter;
ii) be carried out simultaneously with the latter;
iii) carried out after the latter; or
8. Method according to claim 7, characterized in that iv) is carried out before, simultaneously with and after the latter. further comprising activating a magnetic field generator to move the target substance toward an inner wall of the microfluidic channel;
9. The method according to claim 8, wherein the target substance is bound to magnetic particles. (a) increasing the pressure of the gas to transfer the third mixture not bound to magnetic particles and the sample liquid-gas interface from the third zone to i) the second zone; ii) to the first zone, iii) to a capillary stop, or iv) to a sample application port, wherein the target substance bound to the magnetic particles remains within the third zone. , and the process of
(b) simultaneously increasing the pressure of the gas and oscillating the pressure of the gas;
10. The method of claim 9, further comprising: (a) illuminating the third zone with light, thereby causing the optical label to emit a signal;
(b) detecting the signal via an optical detector, thereby indicating the presence of the target substance within the sample liquid;
11. The method of claim 10, further comprising: The step of vibrating the pressure of the gas again, the step of vibrating the pressure of the gas again, or the step of vibrating the pressure of the gas when increasing the pressure of the gas includes: 12. The method of claim 11, including the step of vibrating the corresponding internal spacing between and the second inner wall. The step of vibrating the corresponding internal spacing includes vibration of 75 μm or less, 65 μm or less, 50 μm or less, 40 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, 10 μm or less, 8 μm or less, or 7 μm or less. , less than or equal to 6 μm, at least 1 μm, at least 2 μm, at least 2.5 μm, at least 3 μm, at least 4 μm, at least 5 μm, at least 10 μm, at least 15 μm, or at least 20 μm. changing the corresponding internal spacing over
13. The method of claim 12, wherein the total peak-to-peak distance is measured along an axis perpendicular to a plane defined by the microfluidic device. oscillating the pressure of the gas again, oscillating the pressure of the gas again, or oscillating the pressure of the gas when increasing the pressure of the gas, at a frequency of at least 10 Hz; a frequency of at least 25 Hz, a frequency of at least 100 Hz, a frequency of at least 250 Hz, a frequency of at least 500 Hz, a frequency of at least 700 Hz, a frequency of at least 750 Hz, a frequency of at least 1000 Hz, a frequency of 2000 Hz or less, a frequency of 1500 Hz or less, a frequency of 1250 Hz or less, 12. The method of claim 11, wherein the method is performed at a frequency of 1000 Hz or less, 900 Hz or less, or 800 Hz or less. The steps of reducing the pressure of the gas and oscillating the pressure of the gas include an actuation foot coupled to a contact portion of an exterior surface of the bladder region aligned with at least a portion of the first interior wall. 6. The method of claim 5, wherein the portion of the first inner wall is spaced distally from the sample liquid-gas interface via an actuation system. The portion of the first inner wall may extend from the sample by at least 0.2 cm, at least 0.3 cm, at least 0.5 cm, at least 0.75 cm, at least 1.00 cm, at least 1.25 cm, or at least 1.5 cm. 16. A method according to claim 15, characterized in that it is spaced distally from the liquid-gas interface. The total area of contact between the actuating foot and the contact portion is less than or equal to 12 ^mm2 , less than or equal to 10 ^mm2 , less than or equal to 8 ^mm2 , less than or equal to 6 ^mm2 , less than or equal to 5 ^mm2 , at least 1 ^mm2 , at least 2 ^mm2 , at least 3 ^mm2 , at least 16. A method according to claim 15, characterized in that it is 4 mm ^<2> or at least 5 mm ^<2> . The method is
compressing the internal space prior to introducing the sample liquid into the microfluidic channel;
maintaining the compression of the internal space during introduction of the sample liquid into the microfluidic channel;
6. The method of claim 5, further comprising: Compressing the internal spacing comprises at least 40%, at least 50%, at least 60%, at least 65%, at least 75%, at least 80% of the total internal height of the bladder area as measured before said compression. , reducing the internal height of the bladder region by at least 85% or at least 90%;
The internal height is defined by the distance between the first internal wall and the second internal wall when measured along an axis perpendicular to a plane defined by the microfluidic device. 20. The method of claim 18, characterized in that: compressing the internal spacing comprises reducing the internal height of the bladder region by at least 40 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 75 μm, at least 85 μm or at least 90 μm;
The internal height is defined by the distance between the first internal wall and the second internal wall when measured along an axis perpendicular to a plane defined by the microfluidic device. 20. The method of claim 18, characterized in that: The total internal height of the bladder region defined by the distance between the first inner wall and the second inner wall when measured along an axis perpendicular to a plane defined by the microfluidic device is , 50-200 μm, 75-150 μm, 90-130 μm, or 110 μm before the compressing step. The sample liquid and the sample liquid-gas interface are at least 10 μm/s, at least 20 μm/s, at least 50 μm/s, at least 400 μm/s, at least 600 μm/s, at least 750 μm/s, at least 1000 μm/s, at least 1250 μm /s, at least 1500 μm/s, 2000 μm/s or less, 1900 μm/s or less, 1800 μm/s or less, 1500 μm/s or less, 1250 μm/s or less, 1000 μm/s or less, 750 μm/s or less, 500 μm/s or less, 250 μm/s The method according to claim 1, wherein the method is moved to the first zone at a speed of less than 150 μm/s, less than 100 μm/s, or less than 75 μm/s. A method according to claim 1, characterized in that the volume of the gas that is vibrated ranges from 5 μL to 10 μL, from 6.5 μL to 9.0 μL, or from 6.9 μL to 8.6 μL. The area of the sample liquid-gas interface is at least 0.03 mm ² , at least 0.04 mm ² , at least 0.06 mm ² , at least 0.07 mm ² , at least 0.08 mm ² , not more than 0.25 mm ² , 0.2 mm ² or less, 0.175 mm ² or less, 0.15 mm ² or less, 0.135 mm ² or less, 0.12 mm ² or less, or 0.1 mm ² or less.