MICROFLUID SYSTEM AND METHOD TO TEST FOR TARGET MOLECULES IN A BIOLOGICAL SAMPLE

• US 20100151443A1
• Filed: 12/19/2007
• Published: 06/17/2010
• Est. Priority Date: 12/19/2006
• Status: Active Grant

• Current Assignee: Fio Corporation
• Original Assignee: Fio Corporation
• Inventors: Xiang, Qing, Chan, Warren Che Wor, Klostranec, Jesse M.
• Granted Patent: US 9,360,476 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 33/54346 Nanoparticles
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/587 Nanoparticles
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

A system and method to test for the presence of target molecules in a biological test sample includes test molecules, a microfluidic chip, and irradiating and detection devices. The test molecules include bio-recognition molecules conjugable with the target molecules, and the corresponding conjugates. The microfluidic chip includes sample channels and flow focusing channels adjoining the sample channels. A buffer exiting from the focusing channels directs a single-file stream of the test molecules through one of the sample channels. The irradiating device delivers radiation for absorption by the test molecules in the single-file stream. After absorption, the test molecules emit fluorescence of a distinct fluorescent spectrum for each of the conjugates. The detection device monitors identifies the presence of the conjugates by monitoring for the distinct fluorescent spectrum. Thus, the test system and method identifies the presence of the target molecules in the test sample.

Claims

1. A test system for use with a buffer to test for the presence of target molecules of one or more target types in a biological test sample, the test system comprising:

(a) a first set of detection molecules, each comprising one or more biorecognition molecules (BRMs) immobilized relative to one or more BRM fluorophores, wherein each of the BRMs is specific for one of the target types, so as to form conjugates of the BRMs and the target molecules, if present in the test sample, with the conjugates comprising one or more conjugate types each corresponding to a different one of the detection molecules in conjugation with its specific one of the target types;

(b) a microfluidic chip comprising a chip substrate portion shaped to define;

(i) one or more elongate sample channels therein sized to enable passage therethrough of the conjugates; and

(ii) one or more flow focusing channels therein for operative passage therethrough of the buffer, with the one or more flow^r focusing channels adjoining the one or more elongate sample channels, with the buffer exiting from the flow focusing channels operatively directing a single-file stream of the conjugates through at least one of the sample channels;

(c) an irradiating device operatively delivering electromagnetic frequency (EMF) radiation, at an irradiation position along said at least one of the sample channels, for absorption by the conjugates in the single-file stream, wherein the conjugates emit fluorescence after absorption of the EMF radiation, and wherein the fluorescence of the conjugates comprises a distinct fluorescent spectrum for each one of the conjugate types; and

(d) a detection device monitoring the single-file stream for the fluorescence emitted by the conjugates, wherein the detection device identifies the presence of the conjugates by monitoring for the distinct fluorescent spectrum of each one of the conjugate types;

whereby the test system identifies the presence of the target molecules in the test sample.

2. The test system according to claim 1, wherein each of the detection molecules further comprises a microbead immobilized relative to, and substantially interposed between, the BRMs and the BRM fluorophores, and wherein the BRM fluorophores emit at least a BRM part of the fluorescence of the distinct fluorescent spectrum after absorption of the EMF radiation.

3. The test system according to claim 2, wherein the BRM fluorophores comprise one or more quantum dots, with the quantum dots comprising one or more quantum dot types, and wherein the quantum dots together emit at least said BRM part of the fluorescence of the distinct fluorescent spectrum after absorption of the EMF radiation.

4. The test system according to claim 3, wherein the quantum dots comprise two or more of the quantum dot types.

5. The test system according to claim 2, wherein the BRM fluorophores comprise one or more fluorescent dyes, with the fluorescent dyes comprising one or more fluorescent dye types, and wherein the fluorescent dyes together emit at least said BRM part of the fluorescence of the distinct fluorescent spectrum after absorption of the EMF radiation.

6. The test system according to claim 1, wherein the conjugates are less than about 10 micrometers (μ

m) in size.

7. The test system according to claim 6, wherein the conjugates are less than about 5 μ

m in size.

8. The test system according to claim 7, wherein the conjugates are less than about 1 μ

m in size.

9. The test system according to claim 1, wherein each of the conjugates further comprises a target marker fluorophore bound to a respective one of the target molecules, and wherein the target marker fluorophore emits a target part of the fluorescence of the distinct fluorescent spectrum after absorption of the EMF radiation.

10. The test system according to claim 1, wherein each of the detection molecules further comprises a microbead immobilized relative to, and substantially interposed between, the BRMs and the BRM fluorophores, wherein each of the conjugates further comprises a target marker fluorophore bound to a respective one of the target molecules, and wherein for each of the conjugates, the BRM fluorophores emit a BRM part, and the target marker fluorophore emits a target part, of the fluorescence of the distinct fluorescent spectrum after absorption of the EMF radiation, such that the BRM fluorophores and the target marker fluorophore together emit the fluorescence of the distinct fluorescent spectrum after absorption of the EMF radiation.

11. The test system according to claim 10, wherein the detection device comprises at least two avalanche photodetectors (APDs) monitoring the single-file stream for the fluorescence emitted by the conjugates, with a first one of the APDs adapted to receive and identify the presence of the BRM part, and a second one of the APDs adapted to receive and identify the presence of the target part, of the fluorescence of the distinct fluorescent spectrum for said each of the conjugates.

12. The test system according to claim 11, wherein the target part has a lower intensity than the BRM part of the fluorescence of the distinct fluorescent spectrum for said each of the conjugates, and wherein the second one of the APDs has a greater sensitivity than the first one of the APDs.

14. The test system according to claim 1, wherein said at least one of the sample channels is defined by one or more elongate channel walls of the chip substrate portion, with the channel walls comprising two opposing side channel portions, and wherein the buffer exiting from the flow focusing channels operatively directs the single-file stream of the conjugates along a sample path that is in spaced relation from at least the two opposing side channel portions.

15. The test system according to claim 14, wherein the microfluidic chip further comprises a glass slide underlying the chip substrate portion, with the glass slide defining a bottom channel portion of said at least one of the sample channels, wherein the channel walls further comprise a top channel portion, and wherein the sample path is operatively positioned in said spaced relation from both the bottom channel portion and the top channel portion.

16. The test system according to claim 1, wherein said at least one of the sample channels comprises a sample focused channel, with the sample channels further comprising a sample supply channel in fluid communication with the sample focused channel, and with the sample focused channel being downstream of the flow focusing channels, such that the buffer exiting from the flow focusing channels and the single-file stream of the conjugates operatively flow through the sample focused channel.

17. The test system according to claim 16, wherein a buffer flow rate of the buffer in the sample focused channel is higher than a test flow rate of the conjugates in the single-file stream.

18. The test system according to claim 1, wherein the flow focusing channels comprise at least two flow focusing channels, adjoining the one or more elongate sample channels upstream of said at least one of the sample channels, with the two flow focusing channels adjoining the one or more elongate sample channels from opposing sides of said at least one of the sample channels.

19. The test system according to claim 18, wherein the two flow focusing channels adjoin the one or more elongate sample channels at a common intersection portion.

20. The test system according to claim 19, wherein the buffer exiting from the flow focusing channels operatively focuses the conjugates into the single-file stream by less than about 10 micrometers (μ

m) downstream of the common intersection portion.

21. The test system according to claim 1, wherein each of the one or more flow focusing channels adjoins the one or more elongate sample channels at an adjoining angle of about 90 degrees.

22. The test system according to claim 1, wherein each of the one or more flow focusing channels adjoins the one or more elongate sample channels at an adjoining angle of about 45 degrees.

23. The test system according to claim 1, wherein the chip substrate portion is fabricated from polydimethylsiloxane (PDMS).

24. The test system according to claim 1, wherein passage of the conjugates through said at least one of the sample channels is facilitated by electrokinetic flow.

25. The test system according to claim 24, wherein the flow focusing channels are in fluid communication with the sample channels;

wherein the chip substrate portion is further shaped to define a buffer well adjacent a buffer starting point of each one of the flow focusing channels, a sample well adjacent a sample starting point of the sample channels upstream of the flow focusing channels, and a terminal well adjacent an end point of said at least one of the sample channels downstream of the flow focusing channels;

wherein the test system further comprises a sample well electrode operatively positioned in the sample well, a buffer well electrode operatively positioned in each said buffer well, and a terminal well electrode operatively positioned in the terminal well;

wherein the sample well electrode is operatively supplied with a first electrical potential of a first polarity;

wherein the terminal well electrode is operatively supplied with a second electrical potential of an opposing second polarity; and

wherein each said buffer well electrode is operatively supplied with a third electrical potential of the first polarity.

26. The test system according to claim 25, wherein the third electrical potential is higher than the first electrical potential.

27. The test system according to claim 26, wherein a ratio of the third electrical potential relative to the first electrical potential is about 1.8:

1.

28. The test system according to claim 1, wherein a test flow rate of the conjugates in the single-file stream is at least about 30 conjugates per minute.

29. The test system according to claim 28, wherein the test flow rate is at least about 60 conjugates per minute.

30. The test system according to claim 29, wherein the test flow rate is about 500 conjugates per minute.

32. The test system according to claim 1, wherein the irradiating device comprises a light emitting diode (LED) operatively emitting the EMF radiation for absorption by the conjugates in the single-file stream.

33. The test system according to claim 1, wherein the irradiating device comprises a laser operatively emitting the EMF radiation for absorption by the conjugates in the single-file stream.

34. The test system according to claim 33, wherein the laser has an operating power of between about 2 megawatts (mW) and about 50 megawatts (mW).

35. The test system according to claim 34, wherein the operating power of the laser is between about 20 megawatts (mW) and about 25 megawatts (mW).

36. The test system according to claim 1, wherein the EMF radiation operatively delivered by the irradiating device has an EMF wavelength of about 488 nm.

37. The test system according to claim 1, wherein the detection device comprises three or more avalanche photodetectors (APDs) monitoring the single-file stream for the fluorescence emitted by the conjugates, with each of the APDs adapted to receive and identify the presence of a different range of wavelengths in the fluorescence emitted by the conjugates.

38. The test system according to claim 37, wherein a first one of the APDs is adapted to receive and identify the presence of a green range of wavelengths, wherein a second one of the APDs is adapted to receive and identify the presence of a yellow range of wavelengths, and wherein a third one of the APDs is adapted to receive and identify the presence of a red range of wavelengths.

39. The test system according to claim 37, wherein a first one of the APDs is adapted to receive and identify the presence of a green range of wavelengths, wherein a second one of the APDs is adapted to receive and identify the presence of a yellow range of wavelengths, wherein a third one of the APDs is adapted to receive and identify the presence of an orange range of wavelengths, and wherein a fourth one of the APDs is adapted to receive and identify the presence of a red range of wavelengths.

40. The test system according to claim 37, wherein a first one of the APDs is adapted to receive and identify the presence of a blue range of wavelengths, wherein a second one of the APDs is adapted to receive and identify the presence of a green range of wavelengths, wherein a third one of the APDs is adapted to receive and identify the presence of a yellow range of wavelengths, and wherein a fourth one of the APDs is adapted to receive and identify the presence of a red range of wavelengths.

41. The test system according to claim 1, wherein the detection device comprises a charge-coupled device monitoring the single-file stream for the fluorescence emitted by the conjugates.

42. The test system according to claim 1, wherein the detection device comprises at least two avalanche photodetectors (APDs) monitoring the single-file stream for the fluorescence emitted by the conjugates, with each of the APDs adapted to receive and identify the presence of a different range of wavelengths in the fluorescence emitted by the conjugates;

wherein the detection device further comprises a charge-coupled device monitoring the single-file stream for the fluorescence emitted by the conjugates; and

wherein the detection device still further comprises a switch means for switching between monitoring the single-file stream with either the APDs or the charge-coupled device.

43. The test system according to claim 1, wherein the detection device comprises at least one trip sensor monitoring the single-file stream for the fluorescence emitted by the conjugates, with each said trip sensor generating a digital signal corresponding to an intensity of the fluorescence.

44. The test system according to claim 43, wherein each said trip sensor generates the digital signal only when the intensity of the fluorescence is in excess of a minimum intensity, and wherein each said trip sensor has a different pre-determined said minimum intensity.

45. The test system according to claim 1, further comprising a fiber optic cable, with the fiber optic cable operatively delivering the fluorescence to the detection device from substantially adjacent to the irradiation position along said at least one of the sample channels.

46. The test system according to claim 1, further comprising a housing encasing the irradiating device and the detection device, with the housing sized and adapted for portable and point-of-care diagnostic use.

47. The test system according to claim 46, wherein the housing is sized and adapted for hand-held use.

65. The test system according to claim 1, wherein a buffer flow rate of the buffer in the sample focused channel is higher than a test flow rate of the conjugates in the single-file stream.

66. The test system according to claim 1, wherein the test system is particularly adapted for use with one or more biological test samples selected from the group consisting of blood, urine, sputum, and serum.

67. Use of the test system of claim 1 for a diagnosis of a disease state selected from the group consisting of bacterial disease states, viral disease states, fungal disease states, and vector-induced disease states.

68. Use of the test system of claim 1 for a diagnosis of one or more infectious diseases.

69. Use of the test system of claim 1 for a diagnosis of a condition selected from the group consisting of HIV, HBV and HCV.

70. Use of the test system of claim 1 for a simultaneous diagnosis of two or more of the conditions selected from the group consisting of HIV HBV and HCV.

13. A test system to test for the presence of target molecules of one or more target types in a biological test sample, with the test system being for use with:

a buffer; and

a first set of detection molecules, with each of the detection molecules comprising one or more biorecognition molecules (BRMs) immobilized relative to one or more BRM fluorophores, wherein each of the BRMs is specific for one of the target types, so as to form conjugates of the BRMs and the target molecules, if present in the test sample, with the conjugates comprising one or more conjugate types each corresponding to a different one of the detection molecules in conjugation with its specific one of the target types, with the detection molecules emitting fluorescence after absorption of electromagnetic frequency (EMF) radiation;

wherein the test system comprises;

(a) a microfluidic chip comprising a chip substrate portion shaped to define;

(i) one or more elongate sample channels therein sized to enable passage therethrough of the conjugates; and

(ii) one or more flow focusing channels therein for operative passage therethrough of the buffer, with the one or more flow focusing channels adjoining the one or more elongate sample channels, with the buffer exiting from the flow focusing channels operatively directing a single-file stream of the conjugates through at least one of the sample channels;

(b) an irradiating device operatively delivering the EMF radiation, at an irradiation position along said at least one of the sample channels, for absorption by the conjugates in the single-file stream, wherein the fluorescence of the conjugates comprises a distinct fluorescent spectrum for each one of the conjugate types; and

(c) a detection device monitoring the single-file stream for the fluorescence emitted by the conjugates, wherein the detection device identifies the presence of the conjugates by monitoring for the distinct fluorescent spectrum of each one of the conjugate types;

whereby the test system identifies the presence of the target molecules in the test sample.

31. A test system to test for the presence of target molecules of one or more target types in a biological test sample, with the test system being for use with:

 

a first set of detection molecules, each comprising one or more biorecognition molecules (BRMs) immobilized relative to one or more BRM fluorophores, wherein each of the BRMs is specific for one of the target types, so as to form conjugates of the BRMs and the target molecules, if present in the test sample, with the conjugates comprising one or more conjugate types each corresponding to a different one of the detection molecules in conjugation with its specific one of the target types, with the conjugates emitting fluorescence after absorption of electromagnetic frequency (EMF) radiation; and

 

a microfluidic chip comprising a chip substrate portion shaped to define one or more elongate sample channels therein sized to enable passage therethrough of the conjugates, with a single-file stream of the conjugates passing through at least one of the sample channels;

wherein the test system comprises;

(a) an irradiating device operatively delivering the EMF radiation, at an irradiation position along said at least one of the sample channels, for absorption by the conjugates in the single-file stream, wherein the conjugates emit fluorescence after absorption of the EMF radiation, and wherein the fluorescence of the conjugates comprises a distinct fluorescent spectrum for each one of the conjugate types; and

(b) a detection device monitoring the single-file stream for the fluorescence emitted by the conjugates, wherein the detection device identifies the presence of the conjugates by monitoring for the distinct fluorescent spectrum of each one of the conjugate types;

whereby the test system identifies the presence of the target molecules in the test sample.

48. A test system to test for the presence of target molecules of one or more target types in a biological test sample, with the test system being for use with:

a buffer;

a first set of detection molecules, each comprising one or more biorecognition molecules (BRMs) immobilized relative to one or more BRM fluorophores, wherein each of the BRMs is specific for one of the target types, so as to form conjugates of the BRMs and the target molecules, if present in the test sample, with the conjugates comprising one or more conjugate types each corresponding to a different one of the detection molecules in conjugation with its specific one of the target types; and

an irradiating and detection device capable of delivering electromagnetic frequency (EMF) radiation for absorption by the conjugates, with the conjugates emitting fluorescence after absorption of the EMF radiation, wherein the fluorescence of the conjugates includes a distinct fluorescent spectrum for each one of the conjugate types, and with the irradiation and detection device also capable of monitoring for and identifying the conjugates by the presence of the distinct fluorescent spectrum for each one of the conjugate types;

wherein the test system comprises a microfluidic chip comprising a chip substrate portion shaped to define;

(a) one or more elongate sample channels therein sized to enable passage therethrough of the conjugates; and

(b) one or more flow focusing channels therein for operative passage therethrough of the buffer, with the one or more flow focusing channels adjoining the one or more elongate sample channels, with the buffer exiting from the flow focusing channels operatively directing a single-file stream of the conjugates through at least one of the sample channels;

wherein the microfluidic chip is adapted to operatively receive the EMF radiation from the irradiating and detection device, at an irradiation position along said at least one of the sample channels, for absorption by the conjugates in the single-file stream; and

wherein the microfluidic chip is adapted to enable the irradiation and detection device to monitor the single-file stream for the fluorescence emitted by the conjugates;

whereby the conjugates are operatively identifiable by the presence of the distinct fluorescent spectrum for each one of the conjugate types, such that the presence of the target molecules in the test sample is operatively identifiable by the test system.

49. The test system according to claim 48, wherein said at least one of the sample channels is defined by one or more elongate channel walls of the chip substrate portion, with the channel walls comprising two opposing side channel portions, and wherein the buffer exiting from the flow focusing channels operatively directs the single-file stream of the conjugates along a sample path that is in spaced relation from at least the two opposing side channel portions,

50. The test system according to claim 49, wherein the microfluidic chip further comprises a glass slide underlying the chip substrate portion, with the glass slide defining a bottom channel portion of said at least one of the sample channels, wherein the channel walls further comprise a top channel portion, and wherein the sample path is operatively positioned in said spaced relation from both the bottom channel portion and the top channel portion.

51. The test system according to claim 48, wherein said at least one of the sample channels comprises a sample focused channel, with the sample channels further comprising a sample supply channel in fluid communication with the sample focused channel, and with the sample focused channel being downstream of the flow focusing channels, such that the buffer exiting from the flow focusing channels and the single-file stream of the conjugates operatively flow through the sample focused channel.

52. The test system according to claim 48, wherein the flow focusing channels comprise at least two flow focusing channels, adjoining the one or more elongate sample channels upstream of said at least one of the sample channels, with the two flow focusing channels adjoining the one or more elongate sample channels from opposing sides of said at least one of the sample channels.

53. The test system according to claim 52, wherein the two flow focusing channels adjoin the one or more elongate sample channels at a common intersection portion.

54. The test system according to claim 53, wherein the buffer exiting from the flow focusing channels operatively focuses the conjugates into the single-file stream by less than about 10 micrometers (μ

m) downstream of the common intersection portion.

55. The test system according to claim 48, wherein each of the one or more flow focusing channels adjoins the one or more elongate sample channels at an adjoining angle of about 90 degrees.

56. The test system according to claim 48, wherein each of the one or more flow focusing channels adjoins the one or more elongate sample channels at an adjoining angle of about 45 degrees.

57. The test system according to claim 48, wherein the chip substrate portion is fabricated from polydimethylsiloxane (PDMS).

58. The test system according to claim 48, wherein passage of the conjugates through said at least one of the sample channels is facilitated by electrokinetic flow.

59. The test system according to claim 58, wherein the flow focusing channels are in fluid communication with the sample channels;

wherein the chip substrate portion is further shaped to define a buffer well adjacent a buffer starting point of each one of the flow focusing channels, a sample well adjacent a sample starting point of the sample channels upstream of the flow focusing channels, and a terminal well adjacent an end point of said at least one of the sample channels downstream of the flow focusing channels;

wherein the test system further comprises a sample well electrode operatively positioned in the sample well, a buffer well electrode operatively positioned in each said buffer well, and a terminal well electrode operatively positioned in the terminal well;

wherein the sample well electrode is operatively supplied with a first electrical potential of first polarity;

wherein the terminal well electrode is operatively supplied with a second electrical potential of an opposing second polarity; and

wherein each said buffer well electrode is operatively supplied with a third electrical potential of the first polarity.

60. The test system according to claim 59, wherein the third electrical potential is higher than the first electrical potential.

61. The test system according to claim 60, wherein a ratio of the third electrical potential relative to the first electrical potential is about 1.8:

1.

62. The test system according to claim 61, wherein a test flow rate of the conjugates in the single-file stream is at least about 30 conjugates per minute.

63. The test system according to claim 62, wherein the test flow rate is at least about 60 conjugates per minute.

64. The test system according to claim 63, wherein the test flow rate is about 500 conjugates per minute.

71. A method of facilitating a test for the presence of target molecules of one or more target types in a biological test sample, the method comprising the steps of:

a sample flowing step of passing detection molecules through one or more elongate sample channels formed in a chip substrate portion of a microfluidic chip, wherein each of the detection molecules comprises one or more biorecognition molecules (BRMs) immobilized relative to one or more BRM fluorophores, wherein each of the BRMs is specific for one of the target types, so as to form conjugates of the BRMs and the target molecules, if present in the test sample, with the conjugates comprising one or more conjugate types each corresponding to a different one of the detection molecules in conjugation with its specific one of the target types;

a buffer flowing step of passing a buffer through one or more flow focusing channels formed in the chip substrate portion of the microfluidic chip, with the flow focusing channels adjoining the one or more elongate sample channels; and

a sample focusing step, after the buffer flowing step, of directing a single-file stream of the conjugates through at least one of the sample channels by passage of the buffer from the flow focusing channels into the one or more elongate sample channels.

72. The method according to claim 71, further comprising a conjugate-forming step, before the sample flowing step, of forming the conjugates by introducing the BRMs into the test sample, so as to form the conjugates of the BRMs and the target molecules, if present in the test sample.

73. The method according to claim 72 wherein, in the conjugate-forming step, the conjugates are of less than about 10 micrometers (μ

m) in size.

74. The method according to claim 73 wherein, in the conjugate-forming step, the conjugates are of less than about 5 micrometers (μ

m) in size.

75. The method according to claim 74 wherein, in the conjugate-forming step, the conjugates are of less than about 1 micrometer (μ

m) in size.

76. The method according to claim 72 wherein, in the conjugate-forming step, target marker fluorophores conjugable with one or more of the target types are introduced, such that the target marker fluorophores are immobilized relative to and the conjugates.

77. The method according to claim 71 wherein, in the sample focusing step, the single-file stream of the conjugates is directed along a sample path that is in spaced relation from at least two opposing side channel portions of said at least one of the sample channels.

78. The method according to claim 71 wherein, in the sample focusing step, the single-file stream of the conjugates is directed along a sample path that is in spaced relation from at least top and bottom channel portions of said at least one of the sample channels.

79. The method according to claim 71 wherein, in the sample focusing step, the buffer flows into said at least one of the sample channels at a buffer flow rate that is higher than a test flow rate of the conjugates in the single-file stream.

80. The method according to claim 71 wherein, in the sample focusing step, at least two of the flow focusing channels adjoin the sample channels, from opposing sides thereof, upstream of said at least one of the sample channels.

81. The method according to claim 80 wherein, in the sample focusing step, the two flow focusing channels adjoin the sample channels at a common intersection portion.

82. The method according to claim 71 wherein, in the sample focusing step, each of the one or more flow focusing channels adjoins the sample channels at an adjoining angle of about 90 degrees.

83. The method according to claim 71 wherein, in the sample focusing step, each of the one or more flow focusing channels adjoins the sample channels at an adjoining angle of about 45 degrees.

84. The method according to claim 71 wherein, in the sample focusing step, passage of the single-file stream of the conjugates through said at least one of the sample channels is facilitated by electrokinetic flow.

85. The method according to claim 84, further comprising an electrokinetic step, before the sample focusing step, of supplying (i) a first electrical potential of a first polarity to the sample channels upstream of the flow focusing channels, (ii) a second electrical potential of an opposing second polarity to said at least one of the sample channels downstream of the flow focusing channels, and (iii) a third electrical potential of the first polarity to each one of the flow focusing channels.

86. The method according to claim 85 wherein, in the electrokinetic step, the third electrical potential is higher than the first electrical potential.

87. The method according to claim 86 wherein, in the electrokinetic step, a ratio of the third electrical potential relative to the first electrical potential is about 1.8:

1.

88. The method according to claim 72 wherein the method further comprises the steps of:

an irradiating step, after the sample focusing step, of delivering electromagnetic frequency (EMF) radiation to the conjugates in the single-file stream; and

a fluorescence-detecting step, after the irradiating step, of monitoring the single-file stream for fluorescence emitted by the conjugates, with each of the conjugates, after absorption of the EMF radiation, emitting fluorescence of a distinct fluorescent spectrum for each one of the conjugate types; and

a conjugate-identifying step, after the irradiating step, of monitoring for the distinct fluorescent spectrum of each one of the conjugate types, so as to identify the presence of the target molecules in the test sample.

89. The method according to claim 88 wherein, in the conjugate-forming step, target marker fluorophores are bound to respective ones of the target molecules, such that in the fluorescence-detecting step, the target marker fluorophores emit a target part, and the BRM fluorophores emit a BRM part, of the distinct fluorescent spectrum for each one of the conjugate types.

90. The method according to claim 89 wherein, in the fluorescence-detecting step, fluorescence emitted by the conjugates is received by at least two avalanche photodetectors (APDs), with a first one of the APDs receiving and identifying the presence of the BRM part, and a second one of the APDs receiving and identifying the presence of the target part, of the fluorescence of the distinct fluorescent spectrum for said each of the conjugates.

91. The method according to claim 88 wherein, in the irradiating step, a laser having an operating power of between about 2 megawatts (mW) and about 50 megawatts (mW) delivers the EMF radiation to the conjugates in the single-file stream.

92. The method according to claim 91 wherein, in the irradiating step, the operating power is between about 20 megawatts (mW) and about 25 megawatts (mW).

93. The method according to claim 88 wherein, in the irradiating step, the EMF radiation has an EMF wavelength of about 488 nm.

94. The method according to claim 88 wherein, in the fluorescence-detecting step, the fluorescence emitted by the conjugates is received by a charge-coupled device.

95. The method according to claim 88 wherein, in the fluorescence-detecting step, the fluorescence emitted by the conjugates is selectively received by at least one of a charge-coupled device and one or more avalanche photodetectors (APDs).