WO2006127590A2

WO2006127590A2 - Microfluidic detection cell for stimulated radiation measurements

Info

Publication number: WO2006127590A2
Application number: PCT/US2006/019714
Authority: WO
Inventors: Paul D. Smith; Nicole Y. Morgan; Ed Wellner; Terry M. Phillips
Original assignee: The Government Of The United States As Represented By The Secretary Of The Department Of Health And Human Services
Priority date: 2005-05-20
Filing date: 2006-05-19
Publication date: 2006-11-30
Also published as: WO2006127590A3

Abstract

A microf luidic detection cell (339) for stimulated radiation measurements, such as fluorescence or phosphorescence can have a detection volume of 1 μL or less and a sample can be excited using two excitation wavelengths. The detection cell can include a 5 mm to 10 cm long capillary tube (110, 340) and an excitation fiber (120, 320) proximate to the capillary tube (110, 340) . A detection fiber (150, 345) is also proximate to the capillary tube (110, 340) , and the detection fiber (150, 345) has a diameter the same size or larger than the internal diameter of the capillary tube (110, 340) . A plurality of either or both of excitation (120, 320) and detection fibers (150, 345) can be used.

Description

MINIATURE LASER-INDUCED FLUORESCENCE DETECTOR

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The invention relates to a miniature laser-induced

fluorescence detector having an in-line microfluidic detection cell. The detection cell finds application in High

Performance Liquid Chromatography (HPLC) , Capillary

Electrophoresis (CE) and Mass Spectroscopy (MS) applications,

among others .

Description of the Related Art

[0002] Advanced analytical methodologies are constantly

requiring ever reduced sample sizes in such fields as

neurochemistry, pharmacology, biochemistry, molecular biology,

immunology, trace analysis of drugs in biological samples,

environmental analysis, toxicology, oncology, cosmetology,

food, and forensics . The analytical methodologies include

high-pressure liquid chromatography (HPLC) , capillary

electrophoresis (CE) and mass spectrometry (MS) . A detection

method associated with these methodologies is fluorescence.

[0003] Fluorescence is the emission of a photon by a

molecule following its excitation by an electromagnetic radiation of a precise wavelength. This excitation induces

an energy jump from a fundamental electronic state SO to an

upper electronic energy level Sl. If the molecule comes back

to its fundamental energy level via a radiative process, a

photon is emitted. The photon energy corresponds to the

energy difference between the two levels SO and Sl. Some of

the excitation energy is dissipated by rotational and

vibrational quanta, and so the excitation energy is thus

always greater than the emission energy. Consequently the

emitted fluorescence is always at a longer wavelength than

the exciting light.

[0004] Phosphorescence occurs when the excitation is to a

state that decays by a "prohibited" electronic transition. In

this instance the lifetime of the excited state can be quite

long, and there can be substantial delay between the

excitation event and the emission event.

[0005] Conventional cells for measuring fluorescence are typically unsuited for in-line analysis of small samples.

These cells are typically based on the filling of a cuvette,

followed by the excitation and analysis of a static sample.

[0006] In order to more efficiently measure fluorescence

and reduce sample size, cells have been developed using

capillary tube technology. Figure 1 shows a related art

column capillary detector. Figure 1 shows a laser 10 that generates a laser beam carried by an optical fiber 20 to an

optical bench, where a dichroic mirror 30 reflects the laser

beam to be focused by an objective lens 40 to a cell 50,

which is fed by column capillaries 60. The fluorescence

signal is collected by the same lens 40 and passes through

the dichroic mirror 30. After a series of filters 70, the

signal reaches the photomultiplier tube (PMT) 80, which transforms the fluorescence signal into an electrical signal .

[0007] A capillary detection cell has been proposed by

Chervet et al . (U.S. Patent 5,423,513). This method uses a

bent capillary detection cell in which an external UV/visible

light beam is directed into an elongated section of the

detection cell from a bend thereof. However, this type of

bent capillary detection cell is difficult to manufacture and

therefore expensive. Therefore, this type of cell is not

well suited for use as a disposable cell. Also, this is a

relatively high volume detection cell (up to 20 nanoliters) with a path length of less than 10 mm.

[0008] However, the related art detection cells are hampered by the large required measurement volume . The

related art capillary cells typically have a sample volumes

in the range of 1 μL. However, this large volume is unsuitable for such goals as the measurement of sub-cellular

components such as mitochondrial proteins. For measurement on these types of samples, a sample volume of 10 nL or less

is desirable.

[0009] Another disadvantage of the related art detection

cells arise from the need for frequent cleaning. An

expensive cell must be cleaned and reused. However, an

inexpensive cell can be considered a consumable that can be

thrown away after use .

[0010] An additional disadvantage of the related art technology arises from the reduction in sensitivity as the sample size decreases. Modern research requires detection of

signals from samples at a concentration of 0.5 picogram/mL or

even lower.

[0011] Another disadvantage of the related art technology

is that there are large optical components, such as a

microscope objective, dichroics, filters, or photomultiplier

tubes, which must be positioned adjacent to the detection

cell. These components are required for the coupling of

light into and/or out of the detection cell in the existing

architectures. It is then much more difficult to incorporate

the detection cell into an existing instrument, such as a

mass spectrometer, without substantially increasing the

overall capillary length.

[0012] Accordingly, modern technology requires new detection cells that are easy to construct, inexpensive and provide high sensitivity and utilize very small sample

volumes. For optimal flexibility, these cells should also

have optical fiber coupling from both the excitation and the

emitted light .

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are included to provide a further understanding of the invention and are

incorporated in and constitute a part of this application,

illustrate embodiments of the invention and together with the

description serve to explain the principle of the invention.

[0014] In the drawings:

[0015] Fig. 1 shows a diagram of a related art

fluorescence measurement apparatus; and

[0016] Fig. 2 shows a cross sectional view of a miniature detection cell in accordance with the invention.

[0017] Figure 3 shows a plan view of a bottom piece of a

detection cell in accordance with the invention.

[0018] Figure 4 is a plan view of a top cover (top piece) of the inventive detection cell.

[0019] Figure 5 shows a diagram of a fluorescence

spectrometer incorporating a microfluidic detection cell in accordance with the invention. [0020] Figure 6 is a photograph of the inventive

microfluidic detection cell mounted in a frame.

[0021] Figure 7 is photograph showing a different view of

the mounted microfluidic detection cell.

[0022] Figure 8 shows the laser and photomultiplier tubes of the inventive fluorescence spectrometer.

[0023] Figure 9 shows a fluorescence chromatogram obtained using the inventive detection cell.

[0024] Figures 1OA and 1OB show a plan view and edge view, respectively, of a bottom piece of a second embodiment of a

detection cell in accordance with the invention.

[0025] Figures HA and HB show a plan view and edge view,

respectively, of a top piece of a second embodiment of a

detection cell in accordance with the invention.

[0026] Figure 12 shows a photograph of a second embodiment of a detection cell in accordance with the invention in

assembled form.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Features and advantages of the invention will be

set forth in the description which follows, and in part will

be apparent from the description, or may be learned by

practice of the invention. The objectives and other

advantages of the invention will be realized and attained by the structure particularly pointed out in the written

description and claims hereof as well as the appended

drawings .

[0028] It is to be understood that both the foregoing

general description and the following detailed description of

the invention are exemplary and explanatory of the invention

as claimed. The scope of the invention is defined by the

claims .

[0029] Accordingly, the invention is directed to producing a miniature laser-induced fluorescence detector that

substantially obviates one or more problems due to

limitations and disadvantages of the related art. Any

particular embodiment of the invention might not solve every

problem of the related art described above.

[0030] In one aspect, the invention provides a miniature detection cell comprising a flow cell portion formed from a

capillary tube. The capillary tube portion of the detection

cell can be about 5 mm to 10 cm in length and will typically

have fluidic fittings at each end capable of easy and

repeated insertion into or removal from an existing flow path.

The detection cell can constitute part of a micro-flow

chromatography system, a nano-flow chromatography system or a

capillary electrophoresis system, and the detection cell is

usable as a removable and reinsertable element in systems such as mass spectrometers, electrochemical detectors,

refractive index detectors and ultra-violet detectors.

[0031] The invention, in part, pertains to a detection cell comprising a capillary tube and an "excitation fiber"

having an end proximate to the capillary tube and also a

"detection" or "emission fiber" having an end proximate to

the capillary tube. The excitation fiber (s) couple light

from a source into the capillary tube for exciting a

fluorescent molecule or other light emitting moiety to be

detected. The detection fiber (s) couple emitted light from

the capillary to a photodetector .

[0032] The excitation fiber can have a diameter the same

size or larger than the inner diameter of the capillary tube.

A detection fiber also has an end proximate to the capillary

tube, and the detection fiber has a diameter the same size or

larger than an inner diameter of the capillary tube. Such a

relative diameter provides for maximal collection of emitted

light. By proximate, a distance of 1 mm or less (including

actually touching) is meant. In a preferred embodiment of the

invention, the excitation fiber, the emission fiber and the

capillary are all mutually perpendicular. The excitation and

emission fibers are preferably located at the same cross

sectional plane along the capillary, but together can be

arranged at any appropriate location along the length of the detection cell, provided that an optical window, i.e. a clear

portion of the capillary that can transmit light, is present

at such place on the capillary. (The entire capillary may be

clear, in which case the entire length may serve as an

optical window.) The detection volume is determined by the

volume of the capillary excited with light from the

excitation fiber. The total volume of the flow cell is

determined by the length of the capillary needed for fluidic

connections to a larger system. This length can be from 5 mm

to 10 cm, preferably from 1 cm to 5 cm, and may be as short

as 1 cm to 2 cm. In a preferred embodiment of the invention,

excitation and detection fibers are located at corresponding

locations of the detection cell so that the optimum amount of

emitted fluorescent radiation can be collected by the

detection fiber. That is, the detection fiber is

preferentially located along the capillary in the same cross

sectional vicinity as the excitation fiber, but arranged so

that the numerical aperture of the detection fiber does not

overlap that of the excitation fiber, as is illustrated in

Fig. 2. In a preferred embodiment of the invention, the

excitation fiber and the detection fiber are juxtaposed at

about the same cross sectional plane of the capillary tube

and oriented so that their numerical apertures do not overlap. In this fashion, there is no excitation light directly

incident on face of the detection fiber.

[0033] In the invention, the excitation fiber can have a

numerical aperture the same size or smaller than the

numerical aperture of the detection fiber. The diameter of

the excitation fiber can be the same size or smaller than the

diameter of the detection fiber. The capillary tube can have

a circular or polygonal cross section, preferably, a square

or rectangular cross section. The capillary tube can be

formed from fused silica and may have an inner layer of fused

silica and an outer layer of a polymer such as polyimide . In

the instance of a polyimide-coated capillary, optical windows

are formed at areas where light enters or leaves the

capillary in operation of the excitation fiber and the

detection fiber.

[0034] The detection volume of the flow cell is defined by the volume of the flow capillary that is illuminated by

the excitation light. In the invention, the detection cell

can have a detection volume of about 10 nL or less. The

detection volume can be as small as one nL or even smaller,

e.g. down to 0.25 nL in the instance of a capillary having a

50 μm diameter and a 100 μm diameter excitation fiber.

[0035] The detection cell can be used so that fluorescence

signals at two wavelengths can be simultaneously excited and measured. The excitation fiber can be attached to the

detection cell at about a 90° angle from the detection fiber.

The detection cell can have about a 100 μm by 100 μm inner dimension, that is, a cross section of about 10,000 μm².

[0036] In the invention, optical coupling between the capillary tube, the excitation fiber and the detection fiber

can be optimized by placing an index matching fluid between

the capillary window and the excitation fiber and the

detection fiber. The index matching fluid will reduce or

eliminate changes in refractive index as light traverses the

capillary wall into the detection fiber and/or from the

excitation fiber.

[0037] The invention, in part, pertains to a multi- wavelength detection cell that includes a capillary tube and

at least one excitation fiber having an end proximate to the

capillary tube, where the excitation fiber has a diameter a

same size or larger than an inner diameter of the capillary

tube. One or more detection fibers have an end proximate to

the capillary tube, each with a diameter a same size or

larger than an inner diameter of the capillary tube.

[0038] The detection cell of the invention can be used

with two or more excitation wavelengths. In such

applications, light of each excitation wavelength may be

carried along a separate excitation fiber. Alternatively light at two or more excitation wavelengths can be coupled

into and carried along a single excitation fiber. Also, if

light at two or more emission wavelengths is to be detected,

there can be two or more detection fibers, with each

detection fiber used to detect light at one of the emission

wavelengths. Alternatively, one detection fiber can be used

to detect light at two or more emission wavelengths, by use

of beam splitters and thin-film filters at the far end of the

fiber.

[0039] The invention, in part, also pertains to a method for manufacturing a detection cell . A method for

manufacturing the detection cell of the invention includes

providing a capillary tube having a glass or fused silica

interior layer and a polymer outer layer; removing the

polymer outer layer to form a window to accommodate the

excitation fiber and the detection fiber; bringing an

excitation fiber end proximate to the excitation window; and

bringing a detection fiber end proximate to the detection

window. The excitation fiber typically has a diameter the

same size or larger than the inner diameter of the capillary

tube, and the detection fiber also typically has a diameter a

same size or larger than an inner diameter of the capillary

tube. [0040] Any polymer coating the outside of the capillary

can be removed by heating methods known to the art, to form a

window around the whole or part of the circumference of the

capillary. In a multiple fiber detection system, each

excitation/detection fiber pair would need a separate window.

In some embodiments of the invention multiple detection

fibers may be used to detect a signal excited by one

excitation fiber. Multiple groups of fibers can be used to

provide excitation at multiple wavelengths and detection at

multiple wavelengths .

[0041]

[0042] Reference will now be made in detail to the

preferred embodiments of the invention, examples of which are

illustrated in the accompanying drawings. The figures and

preferred embodiments should not be considered as limiting of the invention; the scope of the invention is defined only by

the claims following.

Figure 2 shows a cross sectional view of a microfluidic

detection cell in accordance with an embodiment of the

invention. The detection cell is formed from a capillary 110.

The capillary 110 is formed from any material that can

transmit visible, infrared and/or ultraviolet (UV) light.

Preferably the capillary 110 is formed from glass or fused

silica and may be coated with a polymer support, such as polyimide. The capillary 110 can have any appropriate shape,

but a circular, rectangular or square cross section is

preferred. The preferable length of the capillary may be

from 5 mm to 10 cm. By microfluidic, one means a cell

capable of handling volumes in the μL range or less. The

invention has the capability to evaluate samples in the range

of 1 nL or less.

[0043] An excitation fiber 120 is brought proximate to the capillary 110. By proximate, from 5 mm to touching is meant.

Preferably the excitation fiber 120 is from 1 mm to touching

the capillary 110. The excitation fiber has a numerical

aperture (indicated by dashed lines 130) sufficient to

encompass the inner diameter 140 of the capillary 110. That

is, the numerical aperture 130 need only be so large as to

irradiate the interior of the capillary cell through which

the sample flows. When the sample is irradiated, the sample

becomes excited and fluoresces or phosphoresces. The emitted radiation is captured by the detection capillary 150. Since

the fluorescence or phosphorescence is emitted in all

directions, the detection fiber 150 should have a numerical

aperture 160 as large as possible so that the maximum amount

of radiation can be detected. In addition, the diameter of

the excitation fiber 120 should be the same size or larger

than the inner diameter of the capillary tube 110, and the diameter of the detection fiber can be the same size or

larger than the inner diameter of the capillary tube 110.

[0044] In order to enhance the efficiency of collection of

the emitted light, mirrors such as 170 can be placed along

surfaces of the capillary tube 110 that are not covered by

excitation or collection fibers. The mirror 170 can be

placed in a holder or made part of the bottom cover 200

and/or the top cover 260. Also, the interiors of the bottom

cover 200 and/or the top cover 260 can be coated with a

mirroring material such as silver. Also, the exterior of the

capillary tube 110 can be coated with a mirroring material.

In another embodiment, mirroring material can be placed

between the fused silica interior layer and the polymer

exterior layer of the capillary tube 110. For optimum

coupling of the emitted light into the fiber 150, curved mirrors may be used.

[0045] In one preferred embodiment of the invention, the

capillary 110 can have an outer dimension of about 363 ± 15

μm with an inner dimension of about 100 μm by 100 μm (± 5 μm) . The excitation fiber 120 can preferably have a diameter of

about 200 μm and a numerical aperture of about 0.22. The detection fiber can preferably have a diameter of about 600

μm and a numerical aperture of about 0.4. The capillary 110

can have different dimensions, typically 50 μm x 50 μm, 75 μm x 75 μm and 100 μm x 100 μm ( (± 5 μm) . Regarding the

excitation fiber, a multimode fiber no larger than 200 μm is preferable, and a numerical aperture of 0.22 is standard.

The emission fiber can be from 100 μm to 1 mm in diameter,

preferably from 350 μm to 1 mm in diameter, with a numerical aperture of from 0.37 to 0.66 being preferred. However, the

invention is not restricted to the aforesaid dimensions, and

any suitable dimensions can be used.

[0046] The capillary 110 can be a square capillary such as those available from Polymicro Technologies. The capillary

can have an OD (outer diameter) of 300 μm, and when coated

with polyimide, have a total OD of 363 + 15 μm. The ID

(inner diameter) of the capillary can be (measured from flat-

to-flat) 50 + 5 μm, 75 + 5 μm or 100 + 5 μm. In a preferred

embodiment of the invention, the 100 μm ID is used. This

dimension can result in a flow-through cell having a 100 μm

by 100 μm (10,000 μm²) inner cross sectional area. However, the capillary can also have a circular or even oval cross

section.

[0047] The capillary tube 110 can be formed from glass

(e.g., borosilicate glass) if visible wavelengths are of

interest. The capillary tube 110 can also be fused silica to

extend the range of wavelengths into the ultraviolet . A

polymer such as polyimide can cover the capillary tube 110. The capillary tube is therefore translucent or opaque.

Therefore, a window must be formed by removing the polymer at

the area where the fibers are to be placed. For multiple

excitation/detection fiber pairs, corresponding multiple

windows are to be formed. The polymer can be burnt off. In

a preferred embodiment of the invention, the polymer can be

burnt off using a heated wire. A nichrome wire heated by an

electric current can be used. Also, a laser can be used to

remove the polymer. The window goes, wholly or partly, around the circumference of the capillary.

[0048] The excitation fiber 120 and the detection fiber 150 can be joined proximate to the glass or fused silica face

of the capillary window. By proximate, a distance from about

1 mm to touching is meant. However, the light transmission

can be enhanced if a drop of water, glycerin or other index

matching fluid is placed between the fiber and the capillary.

Ideally, an immersion fluid that has a refractive index equal to the glass or fused silica in the fiber and/or capillary

should be used. The refractive index of fused silica varies

from 1.40 to 1.55 through the transmission range. While air

has a refractive index of 1.00, room temperature water has a

refractive index of 1.33. Glycerine, with a refractive index

of 1.473, is also a good index matching fluid. In a

preferred embodiment of the invention, enhanced light collection was observed when a drop of liquid (water,

glycerin, index matching fluid or immersion oil) was placed

between the capillary 110, the excitation fiber 120, and the

detection fiber 150. Also, wax or optical cement can be used

to attach the excitation fiber 120 and the detection fiber

150 to the capillary 110. If the surfaces to be joined are

very flat, then optical contacting techniques can be used,

where the capillary and fiber or thoroughly cleaned,

physically joined and then sealed around the edges with a

shellac or cement. However, cementing may negatively impact

the disposability of the cell.

[0049] Also, when water is used as the immersion fluid,

the inventors have observed that a regime of optimized

optical signal is observed as the water evaporates, probably

due to meniscus formation. In this way, a roughly 10-fold

increase in signal was observed.

[0050] In a preferred embodiment of the invention, the excitation fiber 120 and the detection fiber 150 are joined

to a square or rectangular capillary at different faces at an

angle of about 90° to one another. This normal angle arises

naturally from the square geometry of the capillary 110.

However, the invention is not restricted to right angle (90°)

joinder of the excitation fiber 120 and detection fiber 150

to the capillary 110, since the measurement is of light radiation, typically fluorescence, that is emitted in all

directions. Any appropriate angle can therefore be used to

join the excitation fiber 120 and detection fiber 150 to the

capillary 110. Preferably, laser light from the excitation

fiber will not be directly coupled into the detection fiber.

This constraint, coupled with the cell geometry, thus

translates to a range of joinder angle of from about 10° to

about 120°.

[0051] The arrangement of the fibers and the capillary tube can be very versatile. For example, the fibers can be

arranged about a single cross sectional area of the capillary

tube. That is, the excitation fiber and the detection fiber

can be arranged about the capillary tube so that they are

aligned at the same cross sectional plane of the capillary

tube. Also, more than one excitation (or detection) fiber

can be placed at the same cross section. On the other hand,

the fibers can be arranged along the linear length of the

capillary tube. However, for any given excitation/emission

fiber pair, it is preferred that both elements in the pair be

aligned to the same cross-section of the capillary, as is

done in the exemplary holder/device.

[0052] Figure 3 shows a plan view of a bottom piece 200 of

a detection cell in accordance with the invention. The

bottom cover 200 has two grooves 210, 220 cut at right (90°) angles. The two grooves 210, 220 are to hold the excitation

fiber 120 (not shown in the figure 3) and the capillary 110

precisely positioned and at right angles to each other. A

recess 230 defines the area in which the fiber 150 is to be

joined to the capillary 110 and the fiber 120. The recess is

on the opposite side from the grooves, and is present to

provide access for tightening and loosening the sub-miniature

type-A (SMA) coupling of the detection fiber. A circular

opening 240 defines a window into which the detection fiber

150 is to be attached to the bottom cover 200 at an

orientation normal to the surface of plane surface of the

bottom cover 200. The bottom cover 200 also has screw or

bolt holes 250 for fastening the bottom cover 200 to the top

cover 260 so as to provide a holder for the detection cell.

[0053] Although not restricted to these dimensions, the bottom piece 200 can have a length of 1.4 inches. Groove 220,

for holding the capillary 110, can have a dimension of 0.015 inches wide and 0.012 inches deep. Groove 210, for holding

the excitation fiber 120, can be 0.025 inches wide by 0.016

inches deep. This groove should be circular or wedge shaped

so as to hold the excitation fiber precisely in the center of

the groove. The recess 230 can have a diameter of 0.5 inches

and a depth of 0.125 inches. The circular opening 240 can have a radius of 7/32 (0.22) inches. The screw or bolt holes

250 were tapped at 2.5 mm.

[0054] Figure 4 is a plan view of a top cover (top piece) 260 of the inventive detection cell. Groove 270 accommodates

the excitation fiber 120, and allows the center of the

excitation fiber 120 to coincide precisely with the center of

the capillary. An oblong or oval window 280 is formed at the

center of the top cover 260 to avoid compression of the

capillary at the optical window in the coating polymer.

Screw or bolt holes 290 correspond to the screw or bolt holes

250 of the bottom cell. The top cover can have a dimension

of 1.4 inches to a side. The groove 270 can be 1/8 (0.125)

inches wide and .004 inches deep. The screw or bolt holes

290 can be 2.5 mm holes. It is important that the groove 270

be somewhat wider than the excitation fiber 120 so as not to

interfere with the horizontal position already determined by

the position of the groove 210 in the bottom piece 200. The

depth of the grooves are chosen such that the center of the

excitation fiber 120 coincides precisely with the center of

the capillary 110. The fibers will in some embodiments be

mechanically protected, e.g. by a Tefzel^" (DuPont) buffer, and

for practical reasons, the excitation fiber and/or detection

fiber may be larger in outer diameter than the outer diameter of the capillary, or vice versa. The depths of the grooves

are then adjusted accordingly.

[0055] The size of the screw and bolt holes is not important. The overall 1.4 inch dimension is not critical,

but was chosen as a compromise between ease of adjustment

(bigger is easier) and flow cell length (smaller is better) .

[0056] The bottom cover 200 and the top cover 260 of the holder of the inventive detection cell can be milled from

aluminum. Anodizing the aluminum is preferred. However, a

less expensive and thus disposable detection cell and holder

can be constructed from a suitable plastic such as

polypropylene, nylon, polyethylene terephthalate,

polytetrafluoroethylene, etc. Also, the bottom cover 200 and

the top cover 260 can be made by injection molding.

[0057] In practice, the detection cell can be assembled in a highly efficient fashion. The bottom cover 200 and the top

cover 260 can be first bolted together loosely. Then the

capillary tube 110, and the fibers 120, 150 can be inserted

into the channels formed by the matching grooves in the

bottom cover 200 and the top cover 260, and the bolts

tightened to clamp everything in place. To match the

refractive index of the capillary tube with the fibers, the

ends of the fibers, 120, 150 can be wetted with an index

matching fluid (such as water, glycerine or immersion oil) . The application of the index matching fluid is preferably

performed after fiber insertion, through an opening such as

the window 280. Alternately, the capillary tube 110 and the

fibers 120, 150 can be aligned in the corresponding grooves

of the bottom cover 200 before the top cover 260 is bolted on.

[0058] Of course, if a plurality of excitation and/or

detection fibers are used, then the arrangement of grooves

for holding the fibers is modified accordingly.

[0059] Figure 5 shows a diagram of a fluorescence spectrometer incorporating a microfluidic detection cell in

accordance with the invention. An excitation laser 300 emits

laser light for exciting the fluorophore (s) . Any appropriate

wavelength laser can be utilized, such as a 409 nm laser.

Other laser wavelengths can include, but are not restricted

to 266 nm, 308 nm, 330 nm, 375 nm, 405 nm, 470 nm, 532 nm,

633 nm and 660 nm. Also, a tunable laser can be used. The

laser radiation passes through a condensing lens or objective

310 to be coupled into the excitation fiber 320. The

excitation fiber enters the microfluidic detection cell 330

to irradiate the rectangular or square capillary 340.

[0060] The detection fiber 345 carries light from the cell 330 to a collimating lens 350 and through thin film filters

360, which filter out the excitation wavelengths. The

wavelength of the emitted light is dependent upon the particular signaling moiety used and the selection of

appropriate filters and detection fibers would be apparent to

one of ordinary skill in the art.

[0061] The filtered fluorescence signal is then incident

to a photomultiplier tube (PMT) 370 powered by a power supply

380. The electronic signal generated by the PMT is then

processed to produce fluorescence data. Although not shown,

the cell 330 is typically connected in-line with an

analytical device such as a HPLC or CE apparatus.

[0062] The capillary 110 of the microfluidic cell can be

connected to a liquid line using any appropriate fitting or

piping arrangement. Preferably, the connection is made using

a compression-type fitting that minimizes or eliminates any

dead space. The connection can be accomplished using easily

disconnectable microfluidic connectors known to the art, such

as NANOTIGHT or MICROTIGHT connectors from Upchurch

Scientific, Oak Harbor, Washington. However, any suitable

compression fitting or connector can be used.

[0063] Figure 6 is a photograph of the inventive

microfluidic detection cell mounted in a holder or frame.

The capillary can be observed to be entering the left and

right sides of the cell . The excitation fiber can be seen to

be entering the top of the cell. The detection fiber leaves the back of the cell and is not seen. A United States

quarter is seen at the bottom to indicate the scale.

[0064] Figure 7 is photograph showing a different view of the mounted microfluidic detection cell. In this view, the

detection fiber and be seen connected to the rear, i.e.,

bottom piece, of the detection cell. The excitation fiber

enters the detection cell from the top. The light colored

fittings indicate the position of the capillary.

[0065] Figure 8 shows the laser and photomultiplier tubes of the inventive fluorescence spectrometer. At the bottom of

Figure 8, a 409 nm excitation laser is positioned to emit

light through a lens to the excitation fiber. At the top of

Figure 8 is the photomultiplier assembly, which has a

detection fiber, collimating lens, filter and PMT.

[0066] To assemble a detection cell of the invention, a bulkhead sub-miniature type-A (SMA) connector is positioned

(screwed into) tapped hole 240 so that the SMA-terminated

detection fiber will be proximate to the capillary 110

(roughly 20-100 μm distant) . Detection fiber 150 was inserted for positioning, but is then removed, as further

assembly is easier if the holder (bottom piece) 200 can lie

flat on that face. The capillary 110 is cut to length, and an

optical window is made in the capillary (using a heated coil) .

The bottom cover 200 of the aluminum holder (shown in Fig. 3) is already bolted into a black plastic box (shown in Figs 6-

7) . The capillary 110 is threaded through the Microtight

fittings (including compression sleeves used with Microtight

fittings) already in place in the holes in the black box, and

lightly pressed into groove 210 with the optical window

centered over opening 240. The excitation fiber 120 is

cleaved and placed in groove 220. Exact positioning is not

important here, though the end should be close to capillary

110. The top cover 260 (Fig. 4) is placed over the bottom cover 200 and loosely attached to the bottom cover 200 (Fig.

3) using 4 screws through holes 290 and tapped holes 250,

taking care to tighten the four screws evenly to keep plates

parallel. An excitation fiber 120 is slid along groove

210/270 until the end is proximate (20-100 μm distant) to the capillary, as can be seen through the openings 280 and

240. One then checks that the optical window of the

capillary is still in the correct position. Then, the screws

are tightened (evenly) until the fibers can no longer slide.

Microtight fittings (which until now have not been tightened,

and so can slide freely over the capillary) are now tightened

on the capillary per manufacturer's instructions. Set screws

at holes in the black plastic box are tightened to hold the

Microtight fittings in place (mostly to provide strain

relief) . Then, the SMA-terminated detection fiber 150 is screwed into the bulkhead fitting. At this point, the

Microtight fittings (which can be chosen to be unions for

HPLC PEEK tubing) can be attached to the flow path in order

to insert the detection cell into whatever large system is

being used.

[0067] Figures 1OA and 1OB show the bottom plate 400 and

Figures HA and HB show the top plate 460 of one embodiment

of a detector having two excitation fibers and two emission

fibers. Dimensions of the various grooves, screw holes and openings for fittings are similar to those for the single

emission fiber embodiment described above, and the detector

is assembled in a similar fashion.

[0068] In Figures 1OA and 1OB, the groove 410 holds the

capillary 110. The two excitation fibers are laid in grooves

421 and 422. The excitation fibers are brought into

proximity to the capillary; a portion of the exciation fiber

in groove 421 may be unsupported by the groove. The opening

441 is tapped to allow coupling of a SMA fitting or other

fitting holding an emission fiber. The opening 431 is left

open to provide tool access for installing the fitting

holding the emission fiber. The center of the opening 441 is

aligned with the point defined by the crossing of the

centerlines of the grooves 410 and 421. Holes 450 are

provided for inserting a means for connecting the top piece 400 and bottom piece 460 of the detection cell. Holes 451

are optionally provided to allow mounting of the detection

cell onto another item.

[0069] In Figures HA and HB, the holes 490 may tapped so

as to allow tightening of the top and bottom plates together

after the detector is assembled, as described above.

Alternatively, the tightening mechanism may be provided by

the fitting piece itself, e.g. as when a bolt or sliding

collar is used. The opening 442 is made to engage a fitting

holding a second emission fiber in the manner similar to the

opening 441. The center of the opening 442 is aligned with

the point defined by the crossing of the centerlines of the

grooves 410 and 422. Similarly to the hole 431, the opening

432 is made to provide tool access to the fitting inserted

into the opening 442.

[0070] Figure 12 is a photograph of a detection cell of the two excitation fiber, two detection fiber embodiment

described above. The capillary is shown horizontally, and is

clearly seen on the left side; the exit of the capillary is

in shadow. The two excitation fibers can be seen entering

the cell from the top. One of the detection fibers is shown

perpendicular to the plane of the detector. The other

detection fiber is hidden behind the detector. [0071] The optical fibers used in the invention can be

plastic clad silica (PCS) that have a silica core, a silicone

resin optical cladding and a fluoropolymer, such as

ethyltetrafuoroethyene (ETFE) , coating. The core diameter of

the fibers can vary from 200 to 1000 μm (e.g., 200 μm, 300 μm,

600 μm or 100 μm) . Commercial optical fiber technology does make available additional optical fiber choices. For example,

a 400 μm diameter Teflon -clad fiber is available, which is

useful as a detection fiber. Silica-clad silica fibers are

available in 200 μm and 100 μm diameters that are useful as

excitation fibers.

[0072] The numerical aperture of the optical fibers can be

as high as 0.4 or higher. For example a Teflon -clad fiber

may have a numerical aperture as high as 0.66.

[0073] The fiber connectors can be for single mode or multimode fibers. SMA connectors are one of the industry

standard connectors for coupling fibers to instruments or to

each other. Other connectors can be used, such as fiber

channel (FC) or straight tip (ST) connectors. The optical

fibers can be cut and the ends polished using methods known

in the art .

[0074] The invention is not restricted to utilizing excitation by a single laser wavelength. The excitation

fiber can carry two or more excitation wavelengths to _.thus afford a more thorough fluorescence analysis. The downstream

optics can also then include a beam splitter to separately

analyze the fluorescence emissions. Also, more than one

excitation fiber can be used to inject excitation radiation

into the detection cell. Additionally, several of the

inventive detection cells can be placed in line in parallel

or series configurations to permit multiple analyses.

[0075] When more than one wavelength is used, the cell can

take on several configurations. If two excitation

wavelengths are used, both wavelengths can be transmitted

using a single excitation fiber, where instead of a single

laser 300, two lasers are used and their beams made collinear

using mirrors and a dichroic. Alternately, a separate

excitation fiber and laser can be used for each excitation

wavelength. Also, multiple emission wavelengths (which may

arise from multiple fluorophores or phosphors excited at the

same or at different wavelengths) can be measured using the

same detection fiber. Alternately, a different detection

fiber can be used for each emission wavelength. Also, a

mirror or second detection fiber coupled to a single PMT

detector can be placed at the window 280 to improve

sensitivity.

[0076] In an additional embodiment of the invention, multiple, independent detection cells can be used for each wavelength. The detection cells can be placed in-line in

either parallel or series configurations. The low cost and

compact size of the inventive cell makes this option

attractive when analysis of multiple wavelengths is desired.

EXAMPLE

[0077] The inlet adaptor (Upchurch F720 Microtight union)

of an assembled flow cell was attached to a Micro-Tech

Protein 5-C18W-100 micro-bore column via 5-cm of 100- μm internal diameter PEEK tubing. The outlet adaptor (Upchurch F720 union) of the flow cell was attached to a 20-cm length

of identical tubing, which ran to waste. The analytical

column was attached to a Dionex LC Packing Ultimate dual

gradient Micro-flow HPLC and the separation run at 8 μL per

minute. Twenty femtomoles in 1 μL of a commercially available (Sigma/Aldrich Chemical Company) tryptic digest of bovine

serum albumin was injected and the elution monitored on-line

via the detector. The output of the photomultiplier was fed

into a World Precision Instrument Duo-18 data acquisition

interface and recorded on a PC as a virtual recorder. The

chromatogram was screen captured and printed using Corel Draw

7 Software Suite. The resulting chromatogram is shown in Fig.

9.

[0078] The detection cell of the present invention provides for linear detection of analytes, which allows for quantitation of the analyte, at least through the 20 fmole to

500 fmole range in a detection volume of 4 nl .

[0079] Therefore, embodiments of the invention offer many

clear advantages over the conventional art detection cells,

which have high cost, large sampling volumes and insufficient

sensitivity. In contrast, the invention utilizes a low

volume microcapillary and an excitation fiber that has a

diameter and numerical aperture sufficiently small to

illuminate only the interior of the microcapillary, and a detection fiber having a diameter and numerical aperture

larger than that of the excitation fiber. The resulting

inventive detection cell thus is very advantageous for the

analysis of proteins and sub-cellular materials. The

invention has a detection volume as low as 1 nL or less in

comparison to the 2000 nL sample volume typical of the

conventional art. For example, a 1 cm x 50 μm x 50 μm capillary has a detection volume of 250 pL. If a smaller

capillary is used, a smaller diameter optical fiber for

excitation would be needed. The invention therefore offers a

disposable detection cell having one or more advantages of

compactness, sensitivity, ease of manufacture and low cost.

[0080] Also, the inventive detection cell is very

versatile. It can be part of a micro-flow chromatography

system, a nano-flow chromatography system or a capillary electrophoresis system. The inventive detection cell is

usable in combination with detection systems that can be, for

example a mass spectrometer, an electrochemical detector or

an ultraviolet detectors. The detection flow cell would be

connected in-line with the flow path of the other/larger

instrument via the Microtight fittings seen in, e.g., Figs.

6-7. The associated optics and electronics then run

independently, and can be placed several meters away (the

distance limited only by the length of the optical fibers) .

[0081] It will be apparent to those skilled in the art

that various modifications and variations can be made in the

invention as described hereinabove without departing from the

spirit or scope of the invention. It is intended that the

invention covers the modifications and variations of this

invention provided they come within the scope of the appended

claims and their equivalents.

Claims

What Is Claimed Is:

1. A detection cell, comprising: a capillary tube having a length of 5 mm to 10 cm; at least one window in the capillary tube; a fluidic fitting mounted at each end of the capillary tube ; at least one excitation optical fiber having an end proximate to the window of the capillary tube; and at least one detection optical fiber having an end proximate to the window of capillary tube.

2. The detection cell of claim 1, wherein the at least one excitation fiber has a numerical aperture the same size or smaller than a numerical aperture of the detection fiber.

3. The detection cell of claim 1, wherein the diameter of the at least one excitation fiber is the same size or smaller than the diameter of the detection fiber.

4. The detection cell of claim 1, wherein the capillary tube has a circular, square or rectangular internal cross section.

5. The detection cell of claim 1, wherein the capillary tube has a circular, square or rectangular external cross section.

6. The detection cell of claim 1, wherein the capillary tube is formed from fused silica or glass.

7. The detection cell of claim 1, wherein the capillary tube is formed from an inner layer of fused silica and an outer layer of polymer, and optical windows are formed at areas facing the excitation fiber and the detection fiber.

8. The detection cell of claim 7, wherein the polymer is polyimide .

9. The detection cell of claim 1, wherein the capillary has a detection volume of 1 μL or less.

10. The detection cell of claim 1, wherein the capillary has a detection volume of 10 nL or less.

11. The detection cell of claim 1, wherein the at least one excitation fiber is placed proximate to the detection cell at a same cross section of the capillary as at least one paired detection fiber at about a 90° angle from the paired detection fiber.

12. The detection cell of claim 1, wherein the capillary- tube has a cross section of about 10,000 μm².

13. The detection cell of claim 1, wherein optical coupling between the capillary tube, the excitation fiber and the detection fiber is optimized by placing a refractive index matching fluid between the capillary and aperture of the excitation fiber and between the capillary and the aperture of the detection fiber, wherein a refractive index refractive index matching fluid approximates the refractive index of the capillary tube, the refractive index of the excitation fiber and the refractive index of the detection fiber.

14. The detection cell of claim 1, wherein the fluidic fittings are capable of easy and repeated insertion into or removal from an existing flow path.

15. The detection cell of claim 1, wherein the detection cell comprises part of a micro-flow chromatography system, a nano-flow chromatography system or a capillary electrophoresis system, and the detection cell is usable in combination with detection systems selected from the group consisting of mass spectrometer, electrochemical detectors, refractive index detectors and ultra-violet detectors.

16. The detection cell of claim 1, wherein there are a plurality of excitation fibers each having an end proximate to the capillary" tube, each excitation fiber having a diameter a same size or larger than an inner diameter of the capillary tube.

17. The detection cell of claim 16, wherein there are a plurality of detection fibers each having an end proximate to the capillary tube, each detection fiber having a diameter a same size or larger than an internal diameter of the capillary tube .

18. A detection method, comprising: providing the detection cell of claim 1; introducing a liquid sample into the capillary tube; transmitting light of at least one excitation wavelength into the detection cell via the at least one excitation fiber; and measuring emitted light transmitted through the at least one detection fiber.

19. The detection method of claim 18, wherein two or more excitation wavelengths are transmitted along one excitation fiber.

20. The detection method of claim 18, wherein there are a plurality of excitation fibers, and a different wavelength is transmitted along each excitation fiber.

21. The detection method of claim 18, wherein there are two excitation fibers.

22. The detection method of claim 18, wherein there are a plurality of detection fibers.

23. The detection method of claim 19, wherein there are a plurality of detection fibers, each coupled to a detector for detecting emitted light of a different wavelength.

24. The detection method of claim 20, wherein there are a plurality of detection fibers, each paired to a different excitation fiber and each coupled to a detector for detecting emitted light of a different wavelength.

25. The detection method of any one of claims 18-24, in which the emitted light is fluorescent light.

26. A method for manufacturing a detection cell, comprising: providing a 5 mm to 10 cm long capillary tube having a glass or fused silica interior layer and a polymer outer layer; removing the polymer outer layer to form an excitation window and a detection window; bringing an excitation fiber end proximate to the excitation window; and bringing a detection fiber end proximate to the detection window, wherein the detection fiber has a diameter a same size or larger than an internal diameter of the capillary tube.

27. A method for manufacturing a detection cell, comprising : providing a top cover having a groove for a capillary tube having a length of 5 mm to 10 cm, a groove for an excitation fiber and a port for a detection fiber; attaching a bottom cover to the top cover, to form a cavity for the capillary tube and a cavity for the excitation fiber; removing a polymer outer layer from the capillary tube to form a window; inserting the capillary tube into the cavity for the capillary tube; inserting the excitation fiber into the cavity for the excitation fiber so that the excitation fiber has an end proximate to the excitation window; and inserting the detection fiber into the port to bring a detection fiber end proximate to the detection window, wherein the detection fiber has a diameter a same size or larger than an external diameter of the capillary tube.

28. The method according to claim 26, wherein the polymer is removed by heating the polymer.

29. The method according to claim 27, wherein the polymer is removed by heating the polymer.