WO2001050820A1 - Conductive polymer coated nano-electrospray emitter - Google Patents

Abstract

The present invention relates to a nanospray emitter (10) including an emitter body (12) which includes a fluid inlet (14), an outlet orifice (16), and a passage (18) communicating between the fluid inlet and outlet orifice; and an electrically conductive polymer coating (19) on at least a portion of the emitter body. Also disclosed are a nano-electrospray device including the nanospray emitter of the present invention, a method for making a nanospray emitter of the present invention, a method of forming a nanospray using the nanospray emitter of the present invention, and a method of analyzing a solution using the nanospray emitter of the present invention.

Description

CONDUCTIVE POLYMER COATED NANO-ELECTROSPRAY EMITTER

This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/174.548 filed January 5. 2000. which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present in\ ention generally relates to nano-electrospray emitters, nano-electrospray de\ ices containing such emitters, methods of making such emitters, and use thereof.

BACKGROUND OF THE INVENTION

Electrospray ionization ("ESI^"*) (Whitehouse et al.. 1985: Meng et al..

1988) has revolutionized the use of mass spectrometry in bioanalytical chemistry because of its ability to transfer large macromolecules from solution into the gas- phase as intact multiply-charged molecular ions. A special advantage of ESI is the ease with which it may be coupled to liquid chromatography ("LC^") (Banks. 1995), capillary electrophoresis ("CE^") (Smith et al.. 1993). and capillary electrochromatography ("CEC^") (Schmeer et al.. 1994). In the last five years, a number of research groups have developed methods for decreased sample consumption in ESI by using much lower flow rates (nL/min) than with conventional ESI (μL/min) (Emmett and Caprioli, 1994: Kriger et al.. 1995: Valaskovic et al.. 1995a: Kelly et al.. 1997: Wilm and Mann. 1996). Of these low flow ESI methods. the flow rate is controlled by some type of pump in microspray (Emmett and Caprioli. 1994). whereas in nanospray the flow rate is controlled by the potential difference between the emitter and counter-electrode (Wilm and Mann. 1996). Typically, nanospray has been accomplished by pulling silica or glass substrates under heat to produce tapered emitters with small inner diameters, e.g.. a few μm. Attomole level sample consumption has been achieved using nanospray (Valaskovic et al.. 1995a: Valaskovic et al.. 1996a). While nanospray prov ides an a\ enue to achieve low-level detection limits with MS using only a tew μL ot sample. ev en at high salt and/or buffer concentrations, most nanohter-flow ESI emitters suffer from short operating lifetimes, poor durability, and/or low reproducibihtv For example, metallized coatings have been applied to emitter substrates to pro\ ide electrical contact at the ESI outlet, but such emitters are highly susceptible to deterioration

electrical discharge (Valaskovic et al . 1995a)

To overcome this serious limitation, methods to increase metallized emitter lifetime in nanosprav have been developed These include chemical deπvatization ot an organotunctional silane on the emitter substrate prior to metallization (Kπger et al 1995). b> depositing a SιO overcoating atop the metallized la er

ιc et al . 1996b) or

controlled electrochemical deposition oϊ metal film onto the emitter substrate (Kelly et al . 1997) Though these multi-layer and electrolv sis methods have shown improvement in emitter durability for low flow ESI. they are tedious and time-consuming Alternativelv . nonmetal zed emitters have been employed with low flow ESI by remotely coupling the ESI voltage to the emitters (Emmett et al . 1998. Hannis and Muddiman. 1998) A disadv antage of this approach, however, is that it relies on the conductive properties of the solution used, rather than the conductive properties of the emitter itself Solution conditions mav vary widely for both LC and capillary separations, and such emitters ma\ exhibit wide differences in performance depending upon the mobile phase conditions used in the separation Placing a metal wire into the tip for electrical contact has proved beneficial for durabihtv (Kelleher et al . 1997. Cao and Moim. 1997. Fong and Chan. 1999). however it would be preferable to avoid the need to regulate the insertion. removal, or adjustment of an independent metal wire

A.s stated b> Lausecker et al . a good, long-acting and stable electrical contact on the CZE capillary terminus remains the main challenge (Lausecker et al . 1998) in performing MS coupled to capillary separations Thus, to fully exploit the advantages of nanosprav-MS with CE tor the purposes of molecular species identification ot analytes in biological fluids (l e . at sub-picomole levels), a stable and/or reproducible type ot nanosprav emitter must be developed

For nanospray ESI-MS emitters to be useful in coupling to either CE or CEC. the emitters must remain stable throughout the separation process Failure ot the emitter during the course of the separation is not acceptable For quantification in particular, it calibration curves of multiple analytes at multiple concentration levels are to be constructed, single emitters with longer lifetimes or multiple emitters showing reproducible performance and lonization efficiency are needed The present mv ention is directed to ov ercoming these deficiencies in the art

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a nanospray emitter including an emitter body which includes a fluid inlet, an outlet orifice, and a passage communicating between the fluid inlet and outlet orifice, and an electπcallv conductive polvmer coating on at least a portion ot the emitter bodv

A further aspect of the present invention relates to a nano-electrospray dev ice including a counter-electrode and a nanospray emitter of the present invention whose inlet is capable of fluid communication with a fluid source including a fluid containing one or more analytes Either the counter-electrode or the nanospray emitter can be either coupled to a power supply, with the other being grounded, for dev elopment ot a suitable electrical potential between the counter-electrode and the nanospray emitter, which causes the fluid to be drawn through the passage of the nanosprav emitter for discharge from the outlet orifice

Yet another aspect of the present invention relates to a method for making a durable nanosprav emitter This method includes the steps ot providing a nanospray emitter body comprising a fluid inlet, an outlet orifice, and a passage communicating between the fluid inlet and outlet orifice, and casting a thin film of an electπcallv conductive polymer coating onto at least a portion of the nanospray emitter bodv λ still lurther aspect of the present invention relates to a method ot forming a nanospray of a solution which includes passing a solution including an anal te through a nanospray emitter of the present invention under conditions effective to electrically charge the solution in a manner to emit the solution trom the emitter in the torm of a nanosprav Another aspect of the present invention relates to a method of analyzing a solution for an analyte in the solution This method includes passing a solution including an analvte through a nanospray emitter of the present invention under conditions effectiv e to electrically charge the solution in a manner to emit the solution from the emitter in the form of a nanospray. and then analyzing the nanospray emission in a manner suitable to detect an analyte present in the solution The conductive polymer coatings on emitters of the present inv ention are a significant departure from previously existing nanospray emitter coatings, all of which are based on metals The conductive polymer-coated nanospray emitters of the present invention overcome the most problematic limitations of metallized emitters thev provide long-term durability while being simple to coat With respect to a preferred embodiment, polv aniline ("PANI^") coated emitters are desirable because the PANI coating is resistant to corrosion (Wesshng and Posdorter. 1999) while maintaining high mechanical stability and antistatic properties (Tπveldi and Dhawan, 1992) No PANI contamination is observed in the ESI spectra Moreover, in its conductive form PANI is optically transparent (green), allowing for direct viewing of the ESI sample, and possesses outstanding adherence to glass properties (Manohar et al . 1991 ) PANI-coated nanospray emitters are relatively simple to produce and are highly resistant to electrical discharge The sensitivity enhancement of PANI-coated nanosprav emitters is similar to that of gold-coated emitters vs normal ESI tor tested analvtes Because ot the significantly greater durability for the PANI coated emitters ot the present inv ention. the emitters are available for use in analyzing, e g . v CE- MS. highly complex biological matrices including, without limitation, drugs and their metabolites, neuropeptides. neurotransmitters. and biomolecules from complex biological fluids and tissues

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A-B illustrate a PANI-coated nanospray emitter of the present in ention Figure 1A is a longitudinal cross-sectional view of the PANI-coated nanospray emitter, illustrating the relationship between the emitter body and the PANI coating thereon The PANI coating is adjacent to the outlet orifice of the emitter body. Figure IB is an image, prepared by photomicrograph, which illustrates the optical transparency of a water-soluble PANI-coated emitter of the present invention. Figure 2 is a longitudinal cross-sectional view of another embodiment of a PANI-coated nanospray emitter of the present invention. The emitter includes a PANI coating adjacent the inlet to the emitter body.

Figure 3 is a longitudinal cross-sectional view of another embodiment of a PANI-coated nanospray emitter of the present invention. The PANI coating covers substantially the entire length of the emitter body (i.e.. from the inlet to the outlet orifice). Figure 4 illustrates an nano-electrospray device, including a counter- electrode coupled to a power supply and a grounded nanospray emitter whose inlet is in fluid communication with a fluid supply (i.e.. containing a solution to be analyzed by MS).

Figure 5 is a nanospray ESI-Fourier transform mass spectrum ("FTMS") of the polymer mixture PEG- 1000/PDMS- 1250/methy 1-terminated PEG

2000. The symbols that follow represent various polymer species: (Δ) singly- sodiated PEG- 1000. (A) doublv-sodiated PEG- 1000. (★) doublv-sodiated PEG-2000. (-r) triply-sodiated PEG-2000. and (•) singly-sodiated PDMS-1250. Top inset: expansion of m/z region 910-1030. Figure 6 is a nanospray FTMS spectrum of vasoactive intestinal peptide using PANI from xylene dispersions as the coating. Total peptide consumed: 5 femtomoles.

Figure 7A-C are nanospray ESI-FT mass spectra of equine cytochrome c (30 μM). Figure 7A is a normal ESI-FT mass spectrum, sum of 10 scans. Total sample consumed = 17 pmol. Figure 7B is a nanospray ESI-FT mass spectrum using gold-coated emitters, sum of 10 scans. Total sample consumed = 1.3 pmol. Figure 7C is a nanospray ESI-FT mass spectrum using xylene-soluble PANI dispersions coated onto the emitters, sum of 10 scans. Total sample consumed = 680 fmol.

Figures 8A-B are aligned nanospray-FTMS of 29.2 μM myoglobin (49/49/2 methanol/water/glacial acetic acid, v/v/v) using a nanospray emitter that was coated with water-soluble PANI. Qualitativelv'. the same charge states are observed at both 5 min (top. Figure 6A) or 160 mm (bottom. Figure 6B) after the initiation of nanospray. with slightly improved S A^' observed at 160 min

DETAILED DESCRIPTION OF THE INVENTION

One aspect ot the present invention relates to a nanospray emitter which overcomes the previously mentioned deficiencies in the art The nanospray emitter of the present invention includes an emitter body which includes a fluid inlet, an outlet orifice, and a passage communicating between the fluid inlet and outlet orifice, and an electrically conductive polymer coating on at least a portion ot the emitter body

The emitter bodv can be tormed ot anv material suitable tor use in nanospray emitters, preterabl a glass or glass ceramic material Suitable materials include, without limitation borosi cate glasses and glass ceramics, aluminosihcate glasses and glass ceramics, and fused silica glasses

Emitter bodies can be pulled from glass or glass ceramic capillary tubes, forming a tapered (e g . conical) portion of the body which has the outlet orifice at the tip thereof (see Wilm and Mann. 1994) The emitter pulling technique can be performed using a fiber-pulling device ( Sutter Instruments) equipped with either a resistance heating element or a C0₂ laser Instruments equipped with a C0₂ laser are better adapted for use with more substrates (than those equipped with resistance heating elements), only the C0₂ laser is capable of heating tused silica for subsequent pulling It has been shown that this method exhibits reproducible emitter production with respect to taper geometry and orifice inner diameters (Valaskovic et al . 1995b) To open the emitter orifice, if necessary or desired, a number of techniques may be used According to a first approach. HF etching can be perlormed according to known techniques (Valaskovic et al . 1995b) According to another approach, which is perhaps safer to the operator and. therelore. preferred, a micro- pipette beveler (Sutter Instruments, model BV-10) can be employed to grind the emitter ends in a controlled fashion Both approaches enable tailoring of the orifice inner diameter The orifice preferably has an inner diameter of less than about 20 μm. more preferably between about 1 μm to about 5 μm. even more preferably about 3 μm to about 5 μm.

Alternatively, suitable emitter bodies of the type described above can be purchased (in a pre-pulled shape) from various commercial suppliers, including New Objective (Cambridge. MA).

The electrically conductive polymer coating can be any suitable polymer coating which (i) has a conductivity of at least about 1.0 x 10^"J S/cm (at 25°C) and (ii) will resist electrical discharge (i.e.. and remain electrically conductive) under electrospray conditions for a duration of at least about 1 hour. The conductivity of the electrically conductive polymer is preferably between about 1.0 x 10° to about 5 S/'cm (at 25 °C).

The durability of the electrically conductive polymer is dependent upon its longevity under normal electrospray conditions. Preferably, the electrically conductive polymer will resist electrical discharge for at least about 3 to 6 hours. more preferably at least about 24 hours, even more preferably several days or more.

Without being bound by theory, it is believed that suitable electrically conductive polymer coatings of the present invention additionally exhibit electrostatic adherence to the glass or glass ceramic substrate. For example, it is believed that PANI coatings possess positively charged sites within the polymer that interact with negatively charged sites on the glass or glass ceramic substrate.

Although not critical, it is desirable for the thickness of the electrically conductive polymer to be substantially uniform over the entire portion of the emitter body which is coated with the polymer. The thickness of the electrically conductive polymer coating is preferably less than about 10 μm. more preferably between about 2 μm to about 4 μm.

The electrically conductive polymer coating can be applied to any portion of the emitter body which will allow for electrical charge to be conveyed to the solution passing through the emitter body. For example, the electrically conductive polymer coating can be applied to a portion of the emitter body adjacent to the inlet thereof (distal end), a portion of the emitter body adjacent to the outlet orifice thereof (proximal end), or substantially the entire emitter body. Depending on the placement of the coating (proximal vs. distal end), the shape of the nanospray plume can be affected. For example, coating at the proximal end typically yields a narrower nanospray plume whereas coating at the distal end typically yields a broader nanospray plume. Under most circumstances, a narrower plume will be desirable to capture (via the inlet of an analytical device such as a mass spectrometer) a greater volume of the nanospray emission.

One preferred electrically conductive polymer is a polyaniline ("PANI^") coating. PANI is unique amongst electroactive polymers in that its conductive state is achieved by protonation of N in the polymer backbone, as opposed to p-doping and partial oxidation of the polymer π system (Genies et al.. 1990). The analytically pure base form of PANI is given in structure ( I ) below, where the fully reduced state "leucoemeraldine^" corresponds to Y = 1 and the fully oxidized state ^"pernigraniline^" corresponds to Y = 0. When Y = 0.5. PANI is said to be in its "emeraldine^" oxidation state. Treatment of the "emeraldine^" oxidation state of PANI with high acid concentrations (~1 M HC1) leads to the formation of the "emeraldine salt^", in which the oxidized repeat units in structure ( I ) are converted to hydrochloride salt functionalities. It is the emeraldine salt form of PANI that is conductive (MacDiarmid et al.. 1987).

( I )

The PANI coating is characterized by the following properties: high resistance to electrical discharge, with coatings lasting for several days or more under electrospray conditions; a conductivity of about 5 S/cm at 25°C and 1 M HC1 (MacDiarmid et al.. 1987): resistance to corrosion (Wessling and Posdorfer. 1999) while maintaining high mechanical stability and antistatic properties (Triveldi and Dhawan. 1992): optical transparency (green), allowing for direct viewing of the ESI sample; and outstanding adherence to glass properties (Manohar et al.. 1991 ).

One embodiment of the nanospray emitter of the present invention is illustrated in Figure LA. The emitter 10 includes a tapered (i.e.. conical) body 12 ha ing a fluid mlet 14 an outlet orifice 16. and a passage 18 communicating between the fluid mlet and outlet orifice Adhered to a portion of the exterior of the body 12 is a PANI coating 19. which extends to the proximal end containing the outlet orifice 16 As shown in Figure I B. a nanospray emitter of the present invention, which is coated with a water-soluble P ANI coating, is translucent A translucent emitter enables visual examination ot the fluid passing through the passage 18

A second embodiment of the nanospray emitter of the present invention is illustrated in Figure 2 The emitter 20 includes a tapered (l e , conical) bodv 22 having a fluid inlet 24. an outlet orifice 26. and a passage 28 communicating between the fluid inlet and outlet orifice Adhered to a portion of the exterior of the bodv 22 is a P ANI coating 29 which extends to the distal end containing the inlet 24 A third embodiment of the nanospray emitter of the present invention is illustrated in Figure 3 The emitter 30 includes a tapered (I e . conical) body 32 having a fluid inlet 34. an outlet orifice 36. and a passage 38 communicating between the fluid inlet and outlet orifice Adhered to substantially the entire exterior of the body 32 is a PANI coating 39 which extends from the proximal end containing the outlet orifice 36 to the distal end containing the mlet 24

A further aspect of the present invention relates to a method of making a nanospray emitter ot the present invention Basically, the method is carried out by providing a nanospray emitter body which includes a fluid inlet, an outlet orifice, and a passage communicating between the fluid inlet and outlet orifice and then casting a thin film of an electπcallv conductive polymer coating onto at least a portion ot the nanospray emitter bodv

The process step of casting can be achieved by a number of procedures

According to a first approach, application of the conductive polymer coating can be carried out by dipping the portion of the emitter body into a soluble suspension which includes the conductive polymer The soluble suspension can be either an aqueous suspension or in an organic solvent After dipping, the solvent is allowed to evaporate, leaving behind the PANI coating on the portion of the emitter body dipped into the suspension

When aqueous suspensions are employed, the PANI is preferably deπvatized to ensure its solubihtv in water A water soluble PANI derivative (Chen and Hwang. 1995) is commercially av ailable from Monsanto (St Louis. MO) Although PANI films cast from aqueous suspensions are susceptible to dissolution by some protic solvents, such PANI films are still functional for nanospray

When organic suspensions are employed, a suitable organic solvent should be selected on the basis of its abihtv to form a soluble suspension of P ANI without degradation of the quahtv ot P ANI film produced upon evaporation of the solv ent Suitable organic solvents include, without limitation, xvlene and a mixture of xv lene and a diacetone alcohol (e g . 4-hy droxy-4-methy l-2-pentanone). typically in an approximate 1 1 ratio Unlike P A I films cast from aqueous solutions. PANI films cast from xylene solutions are not susceptible to degradation by exposure to protic solvents To preclude tackiness ot the resulting PANI film, not more than about 5% by weight of PANI should be suspended in an organic solvent

To ensure that the film resulting from the dipping process does not interfere with the outlet orifice, this first approach may also include the step of passing a fluid through the passage of the emitter body during the dipping step and, optionally, during the curing step The fluid is preferably a gas. such as air. or a non- reactive gas. such as nitrogen

To control the rate of deposition of the conductive polymer onto the emitter body (and. thus, control the thickness of the conductive polymer and its uniformity along the length of the emitter body), it may be desirable to utilize a device

(e a lab jack) which can be used to lower and raise the emitter body into the suspension containing the conductive polymer at a suitable rate For PANI suspensions, the device can be used to ithdraw the emitter body from the suspension at a rate of about 5 mm/s This should achieve a PANI coating of suitable thickness According to a second approach, the step of casting can be carried out by polymerizing the conductive polymer onto the portion of the emitter body in an acidic solution by oxidation By way of example, oxidative polymerization of aniline (0 10 M) can be carried out in aqueous solution by

(ammonium peroxvdisulfate 0 1 M) treatment at -0-5 °C in 1 M aqueous HC1. as described in earlier protocols (Chiang et al . 1986. MacDiarmid. 1987) Preferably, a vanadium oxiαe catalyst is utilized to afford consistent results from the polymerized films (MacDiarmid. 1987) A portion of the emitter body is dipped into the catalyzed solution to poly merize the film on the portion in the solution The time length in which the emitter body is present in the catalyzed solution will affect the thickness of the resulting polymerized film Typically, a time length of about 5 to about 60 minutes is sufficient, more preferably about 15 to about 20 minutes The thicknesses of the electrically conductiv e film should be as set forth above Upon remov al of the emitter body from the catalyzed solution polv meπzation can be quenched by washing the emitters with aqueous NH₄OH (Chiang et al 1986. Fong and Schlenoff. 1995) Afterward, the emitters can be washed with 1 M HC1 to ensure the film is in its conductive state, as indicated v lsually (for P ANI. by a green film) As noted above, a gas can be blown through the passage of the emitter bodv to prev ent clogging of the outlet orifice by the polymer

According to a third approach, the step of casting can be carried out by coating the portion of the emitter body in a solution containing a non-conductive base form of the conductive polymer, and then doping the coated emitter to produce a conductive polymer coating By way of example. PANI film can be formed out of N- methy 1-2-pyrrolιdιnone solution using commercially available PANI (Polysciences. Inc . \\ arrington. PA) Conductive PANI is immersed into basic solution (pH -8) for several hours to produce the non-conductive emeraldine base form (structure I), which is soluble in N-methyl-2-pyrrolιdιnone The N-methyl-2-pyrrohdιnone is then doped with 1 0 M HC1 (pH ~0) to produce the conductive form of PANI. or emeraldine salt " The emitter substrate is dipped into the doped solution, then remov ed and dried This coating procedure produces optically transparent PANI films that conduct electricity

As noted above, a gas can be blown through the passage of the emitter bodv to prev ent clogging of the outlet orifice by the polymer

According to a fourth approach, the step of casting can be carried out by introducing the emitter body, or portion thereof, into an RF discharge chamber and then exposing the emitter body, or portion thereof, to aniline monomers under conditions effective to coat the portion of the emitter body with polyanihne By way of example, the emitter body can be introduced into an RF discharge chamber (Vargo et al , 1989, Vargo et al . 1991 ) along with aniline apor (a few tenths of a torr) and exposed to RF discharge (about 50-100 kHz RF discharge frequency at about 10-50W ) to deposit an electπcallv conductive PANI film on the surface of the emitter body (Bhat and Joshi. 1994. Cruz et al.. 1997. Gong et al., 1998). Interestingly . Gong et al showed that the physical and chemical properties of PANI films could be changed by altering the rf-plasma discharge conditions (Gong et al . 1998) With increasing discharge duration and rf-discharge power, the relative percentage of quinoid groups (relative to benzenoid) was increased, as was the degree of aliphatic cross-linking (Gong et al . 1998) However, the relative percentage of free radicals as found to be higher when lower rf-plasma powers were employed Optionally , to enhance conductivity, the reaction chamber can be doped with iodine vapor (Bhat and Joshi. 1994) or the relative humidity can be increased (Cruz et al., 1997)

Hav ing prepared an emitter of the present invention, it is intended to be used in a nano-electrospray device for creation of low sample volume nanosprays Therefore, a further aspect of the present invention relates to a nano-electrospray device which includes a nanospray emitter of the present invention, as described above The nano-electrospray device 40, illustrated in Figure 4. includes a grounded emitter 10 of the present invention and a counter-electrode 42 connected to a power supply 44 The emitter 10 is supported by an emitter mount 44 such that the emitter can be coupled in fluid communication with a fluid source 50. which holds a fluid (containing one or more analytes) to be examined During use of the nano- electrospray device, the solution delivered to the tip of the emitter body experiences the electric field associated with the maintenance of the tip at high potential Assuming a positive potential, for example, positive ions in solution ( I e . in a condensed phase) will accumulate at the surface, which is drawn out in a downfield direction to establish a Taylor cone At a high enough imposed electric field, the cone is drawn to a filament which produces positively charged droplets via a budding process when the surface tension is exceeded by the applied electrostatic force As solution at the tip is dispersed into a gas phase (of the nanospray). additional fluid is drawn to the tip via capillary action Thus, development of a suitable electrical potential between the counter-electrode 42 and the emitter 10 (I e . through the electrically conductive polymeric coating on the emitter) causes the fluid to be drawn through the passage of the emitter for discharge from the outlet orifice by establishing an electrical field force which exceeds the surface tension of the fluid Although Figure 4 shows the emitter being grounded and the counter- electrode connected to the power supply, it should be appreciated by one of skill in the art that a suitable electrical potential can also be activated by grounding the counter-electrode and coupling the emitter to the power supply. The nano-electrospray device 40 is intended to be used in conjunction with any one of the following devices: a mass spectrometer, whose inlet can be coupled to receive the nanospray from the outlet orifice of the nano-electrospray device 40: liquid chromatography columns, whose outlet can be coupled to the inlet of the emitter on the nano-electrospray device 40: capillary electrophoresis columns, whose outlet can be coupled to the inlet of the emitter on the nano-electrospray device

40: and capillary electrochromatography columns, whose outlet can be coupled to the inlet of the emitter on the nano-electrospray device 40. One of skill in the art may readily appreciate that other equipment can readily be coupled to the nano- electrospray device 40 to introduce a fluid to be analyzed into the inlet thereof or to receive a nanospray from the outlet orifice thereof.

Thus, a further aspect of the present invention relates to a method of analyzing a solution for an analyte in the solution. This method is carried out by passing a solution including an analyte through a nanospray emitter of the present invention under conditions effective to electrically charge the solution in a manner to emit the solution from the emitter in the form of a nanospray. and then analyzing the nanospray emission in a manner suitable to detect an analyte present in the solution. Basically, the nanospray emission can be analyzed using now known or hereafter developed devices which are suitable for the intended analysis to be performed. For example, without being limited thereby, a mass spectrometer can receive the nanospray emission to identify one or more analytes in the nanospray emission.

With the use of a nano-electrospray device of the present invention, it is possible to determine the pharmacokinetic behavior of pharmaceutical agents, the behavior of endogenous biochemicals (e.g.. neuropeptides, neurotransmitters. nucleic acids, proteins, other intercellular signaling molecules, etc.). or the interaction of pharmaceutical agents with endogenous biochemicals in the formation of drug complexes (e.g.. drug-protein complexes). EXAMPLES

The following examples are intended to be illustrative of the present invention, but are no means intended to the limit the scope thereof

Materials & Instrumentation:

The following materials were used in the Examples provided below water-soluble conducti e PANI dispersions (5% aqueous, conductive torm) was obtained from Monsanto (St Louis. MO), where the water-soluble PANI is tunctionahzed with proprietary tethers and is otherwise commercially available from

Monsanto as PANAQUA (without the tethers. PANI is not soluble in water (Chen. 1995 )). xv lene-soluble conducti e PANI dispersions (M 1 17.000 Da. 48 16% \y lenes. 12 62 % butyl cellosolv e. the remainder solid PANI) was obtained from Monsanto (St Louis, MO). polyethylene(glycol) (M_n 1000. PEG- 1000), poly(dιmethylsιloxane) (M_n 1250. PDMS-1250). and equine cy tochrome c were obtained from Sigma, and methyl-terminated poly ethylene(glycol) (M„ 2000. ME PEG-2000) was obtained from Aldπch (Milwaukee. WI) Electrospray lomzation-mass spectra analy sis were performed using the following instrumentation

All ESI mass spectra were acquired using a 3 tesla Bruker Daltonics Bio Apex 30es (Billerica. MA) Fourier transform mass spectrometer equipped with an Analytica of Brantord (Branford. CT) nanospray source A metal cap (250 μm) was fitted over the capillary inlet to the mass spectrometer to reduce the diameter of the opening and to decrease the probability of electrical discharge stripping the conductive coating off the mass spectrometer^'s inlet capillary , making it functionally useless PANI-coated nanospray emitters were inserted into the emitter mount (Fig 4) and loaded with sample by the following means A gas-tight Hamilton syringe containing the sample was connected to a union inlet by 1 5 cm length of fluorinated ethy lene polymer (FEP) tubing ( I T 6^" OD. 0 03^"' ID) A 3 cm segment of Poly micro Technologies (Phoenix. AZ) fused silica tubing ( 186 um OD. 50 μm ID) connected to the outlet The silica tubing was then inserted into the P \NI-coated emitter to its taper, and sample was slowly injected using the syringe. In this way. the liquid analyte forms a plug without bubbles in the emitter tip. The PANI-coated emitter was held at ground and positioned to -0.5-2 mm distance from the metal-capped capillary inlet to the mass spectrometer (as viewed from a microscope mounted on the nanospray source) using the micrometer on the nanospray source. The onset of nanospray was initiated with the counter-electrode at —650 V. and spray was conducted typically at -650 to -1200 V. Ions were accumulated in the external hexapole (Lau et al.. 1997) and electrostatically injected into the Infinity cell (Caravatti and Allemann, 1991 ) (2.0 V trapping potential) using Sidekick (U.S. Patent No. 4.924.089 to Caravatti). Frequency-sweep excitation from m/z 294 to 2400 was applied at an amplitude of -30 V_pp. Detection was in direct mode (bandwidth 0- 156250 Hz). The data sets were apodized with a Gaussian function. Fourier transformed, and displayed in magnitude mode using XMASS 4.0.2 on a Silicon Graphics (Mountain View. CA) RPC 4600 INDY data station.

Example 1 - Preparation of Emitters Coated with Water-soluble PANI

Gold-coated and uncoated borosilicate glass emitters (1.2 mm OD, 0.69 mm ID) pulled to tapers for the ESI outlet with 4±1 μm ID were purchased directly from New Objective (Cambridge. MA). The uncoated emitters were gently swiped using the sealed end of a melting tube upon which aqueous conductive PANI had been applied while the emitter was rotated manually. Though this application method does not provide a PANI coating of uniform thickness, it is easy to confirm by visual inspection whether or not the ESI outlet is covered by a transparent green coating of PANI. A very low flow of air could be (but was not always) run through the emitter to prevent clogging of the emitter orifice during application of water- soluble PANI. The PANI-coated emitter was then allowed to air dry. Within three minutes, the PANI coating was sufficiently adherent to the substrate such that the emitter was usable for nanospray. Photomicrographs (Figure IB) were obtained using a Nikon Optiphot confocal microscope in the Confocal Microscopy and 3-D Imaging Lab in the School of Medicine and Biomedical Sciences at SUNY-Buffalo. Example 2 - Performance of Emitters Coated with Water-soluble PANI

Nanospray was initiated with PANI-coated emitters when the counter- electrode voltage was between -600 and -1200 V. Because of the transparent green PANI coating over the emitter substrate (Figure IB), one could determine whether any air bubbles were present that might interfere with nanospray using a microscope. An example of nanospray (-40 nL/min flow rate) using the water-soluble PANI- coated emitters is shown in Figure 5. which is a mixture of three synthetic polymers dissolved in methanol:2-propanol: 10 mM NaOH (49:49:2. v/v/v): methyl-terminated PDMS (M_n 1250). PEG (M_n 1000). and methyl-terminated PEG (M„ 2000). The

NaOH was added to provide a cation source for creating PDMS ions. Singly- and doubly-charged oligomer ions of PEG- 1000 and doubly- and triply-charged oligomer ions of methyl-terminated PEG-2000 are observed, as are singly-sodiated oligomer ions of PDMS. Similar results were obtained when xylene-soluble PANI was used. Interestingly, normal flow ESI (i.e.. no nanospray) of this same mixture reveals only the PEG distributions, and the PDMS is absent. In distinguishing it from nanospray. normal flow ESI is defined here as being performed with a metal needle, (grounded relative to counter-electrode held at -3 kV). 50 μm i.d.. at 1 μL/min flow rate. Perhaps the higher surface area-to-liquid volume ratio of nanospray vs. normal ESI enables ionization and detection of the more hydrophobic

PDMS. No evidence for any ion derived from PANI is found in the spectrum. Thus. PANI-coated nanospray emitters can be used without the worry of possible contamination from the PANI. In spite of the spectral complexity of the data for the three polymers in Figure 5. the high mass resolving power of FTMS allows for easy identification of charge state (top inset) (Henry. 1991 ). while the ionic species^" identities can be confirmed by high mass accuracy (Maziarz et al.. 1999).

The PANI-coated emitter used for the sample in Figure 5 maintained continuous spray for 40+ minutes before sample was exhausted. Inspection of this emitter under a microscope did not indicate any clear signs of degradation. This was actually somewhat surprising, since it was observed that PANI cast from water- soluble dispersions will dissolve in protic solvents (methanol. ethanol. and water), though such films are exceedingly stable in the presence of 2-propanol and acetonitrile. Of course, the fluid never comes in direct contact with the PANI film (save perhaps at the very edge of the tip). Thus, while some dissolution of the film might be expected if a protic solvent comes into direct contact with PANI cast from water-soluble dispersions, this has not yet prevented the tips from being durable. A 50/50 mixture of methanol and 2-proponal was injected into the emitter and spray was reinitiated to rinse the emitter tip. The next day. this same emitter was used to collect nanospray mass spectra for numerous samples, with rinsing in-between. Even after running multiple samples over two days through the same PANI-coated emitter, stable signal could be obtained without electrical discharge and the emitter continued to be functional. Indeed, with rinsing in between sample loadings, the PANI-coated emitters can be reused many times. While such capability may not be needed for general sample analysis, the availability of stable emitters for coupling to sheathless CE-MS is still an analytical challenge (Lausecker et al.. 1998). Thus, the current PANI-coated emitters may prove useful in coupling nanospray to CE-MS. The emitters are extraordinarily stable to electrical discharge. Under normal operating conditions, no evidence of electrical arcing was observed (either by destruction of the PANI coating or from direct observation of the PANI tip). Arcing could only be induced when the counter-electrode voltage was set above -3 kV and when the tip was placed within 0.2 mm of the counter-electrode (indeed, arcing could only be guaranteed if the tip was inserted directly into the inlet hole in the counter- electrode). Gold-coated tips could be induced to arc at much lower voltages ( — 1 kV) even 2 mm away from the counter-electrode.

Example 3 - Preparation of Emitters with Xylene-soluble PANI

Gold-coated and uncoated borosilicate glass emitters (1.2 mm OD, 0.69 mm ID) pulled to tapers for the ESI outlet with 4±1 μm ID were purchased directly from New Objective (Cambridge. MA). The uncoated emitters were gently swiped using the sealed end of a melting tube upon which xvlene conductive PANI had been applied while the emitter was rotated manually. The xvlene conductive PANI suspension obtained from Monsanto (St. Louis. MO) was modified to reduce the PANI concentration to not more than about 5% bv weight. Though this application method does not provide a PANI coating of uniform thickness, it is easy to confirm by visual inspection whether or not the ESI outlet is covered by a transparent green coating of PANI. A very low flow of air could be (but was not always) run through the emitter to prevent clogging of the emitter orifice during application of xylene-soluble PANI. The PANI-coated emitter was then allowed to air dry. Within three minutes, the PANI coating was sufficiently adherent to the substrate such that the emitter was usable for nanospray.

Example 4 - Performance of Emitters Coated with Xylene-soluble PANI

As noted previously . PANI has high mechanical stability and antistatic properties (Trivedi and Dhavvan. 1992). Part of the stability imparted by the PANI coating may be due to its thickness. PANI films cast from xylene-soluble dispersions, however, are insensitive to degradation by protic solvents. An example of the routine low detection limits achievable with PANI- coated nanospray emitters using an FTMS detector is shown in Figure 6. Here, vasoactive intestinal peptide (VIP) ( 1 ng/μL in methanol/water/acetic acid 49/49/2) was electrospraved at -40 nL/min over 25 sec using a borosilicate nanospray emitter coated with a xylene-soluble dispersion of PANI. Thus. -17 nL sample volume, or 17 picograms (5 femtomoles) of total peptide were used to acquire the ESI-FTMS spectrum in Figure 6. Flow rate was determined by injecting 5 μL of sample and waiting until tip stops producing signal: for the tip used in Figure 6. -120 min.

Use of PANI films cast from xylene-soluble dispersions with nanospray is shown in Figure 7. Fig. 7a is an ESI-FT mass spectrum of equine cytochrome c (30 μM. 50/50 v/v methanol/water) using conventional ESI (flow rate =

1 μL/min). The spectrum shown in Fig. 7b is nanospray of an identical cytochrome c solution using instead gold-coated nanospray emitters (flow rate -100 nL/min). Using nanospray emitters coated with xylene-soluble PANI. the same sample ( flow rate -40 nL/min) generates the spectrum observed in Fig. 7c. The normal ESI spectrum reveals a multiplicity of charge states from 6+ to 15-. while the nanospray spectra reveal charge states over the range 6+ to 1 1+. The discrepancy between charge state distributions for cytochrome c using conventional ESI and nanospray is not inherently obvious One possibility is that ions are desolvated more quickly in nanospray than in ESI. thus ions spend more time in the "gas-phase^"" if formed from nanospray Perhaps this results in increased charge stripping Of course, another possibility is that conformational effects may limit charging ot a protein, though it is not immediately clear why conformation should be any different for ions produced by nanospray vs ESI

One of the key attributes of low flow ESI that has been noted is its high sensitivity (Emmett and Caprioli. 1994. Kπger et al . 1995. Valaskovic et al . 1995a. Kelly et al . 1997 dm and Mann. 1996. Valaskovic et al . 1996a) The total amount of cytochrome c consumed to acquire the spectrum in Fig 7a was 17 pmol. while onlv 1 3 pmol was required to obtain the spectrum in Fig 7b (using gold-coated nanospray emitters) and 680 fmol to obtain the spectrum in Fig 7c (using PANI- coated nanospray emitters) Thus. PANI-coated nanospray emitters show improved sensitivity (as do gold-coated emitters) vs normal flow ESI While the total signal and signal-to-noise ratios are superior with the PANI-coated emitter in Fig 7c v s the gold-coated emitter in Fig 7b. it is likely this is due to the flow rate differences used to acquire the two spectra

Example 5 - Stability of PANI Films on Emitters

The durability of the PANI films for nanospray is also made ev ident in Figure 8 Here, the same nanospray emitter is used to acquire nanospray-FT mass spectra of myoglobin over 160 minutes In comparison to convention gold-coated emitters, the PANI coated emitters of the present invention were useful after far longer than the typical 15-30 minutes sur ival on the ESI-FTMS equipment utilized herein (which is consistent with their reported survival. Valaskovic et al . 1995a) Thus this demonstration illustrates that nanospray emitters with PANI coatings have similar sensitiv ity yet superior durability to gold-coated emitters In addition, several types of cast PANI films exhibit solvent stability

As shown in Table 1 below, in general PANI films cast from water-soluble dispersions ha e much lower stability in protic solvents than those cast from xy lene- soluble films While degradation of the films bv the naked eve is often difficult to detect, inspection v ia microscopy shows that solvent exposure can. in fact, dissolve PANI coatings at the emitter orifice in most cases However, even when PANI films do dissolve upon solv ent exposure, the PANI-coated nanospray emitters were still operable over a period of days, and could be reused many times Films produced from PANI dispersions in xvlene (Monsanto. Inc ) show stability to protic solv ents, but prove to be ery sticky at concentrations abov e 5% PANI by weight

Table 1 Solv ent Stabilities of PANI Films

Solvent Water-soiuble PANI Xvlene-solubie PANI water dissolves immediately stable over 24 h isopropanol Stable tor 10 m slightly soluble Slow dissolution complete after 20 h within 24 h ethanol Forms suspension in minutes, Stable over 24 h complete dissolution after 20 h methanol forms suspension in minutes Stable ovei 24 h acetonitπle Stable over 20 h Stable over 24 h glacial acetic acid Stable for 10 mm . but dissolves Stable over 24 h completely within 20 h nitric acid (1 M) dissolves immediately Slight dissolution within 24 h sodium hydroxide PANI film turns blue, dissolves stable after 24 h . no color

( 1 15 M) slowly, and will not produce stable chanεe

ESI even if emitter is removed from solvent before dissolution

DMSO dissolves immediatelv Stable over 24 h

Example 6 - Electrospray Ionization-Mass Spectral Analysis of Paclitaxel and

Proposed Use of Nanospray

ESI-LC/MS has proved valuable for drug development because of its ability to provide structural information and quantify the drug and its metabolites with lower detection limits than UV spectroscopy (Lee et al . 1997) For example, paclitaxel (taxol ) is an alkaloid that has received considerable attention because of its efficacy in treating ovarian tumors (Suffness. 1995) Paclitaxel promotes tubulin polymerization and inhibits depolymeπzation. thereby inhibiting cell division in cancer cells (Parness et al . 1982) A sharpened picture of the mechanistic and metabolic pathways of paclitaxel may lead to improvements in its clinical efficacy (Dorr. 1997) Thus, in spite of FDA approval, further review of pachtaxel's biotransformation is still needed An HPLC assay of paclitaxel in mouse plasma using UV detection was developed by Sharma et al. (Sharma et al.. 1994). This method can quantify paclitaxel for up to 4-8 h after I.V. injection. Adequate description of the pharmacokinetics requires quantification over a 24-36 h period. Such information from test subjects is currently unavailable. Information of this type would prove useful in determining dosage requirements and treatment frequency for cancer patients using paclitaxel in clinical trials.

ESI-MS and ionspray-MS have been used for structural characterization of paclitaxel and its metabolites (Poon et al.. 1996: Volk et al.. 1997: Lee et al.. 1997: Kerns et al.. 1995: Blay et al.. 1993: Bitsch et al.. 1993). The following ESI-MS protocol shall be used to analyze paclitaxel in mouse plasma samples. Paclitaxel is ESI stable in an 80% acetonitrile:20% ammonium acetate buffer solution at pH 3-7 (pH adjusted by adding acetic acid), and the presence of both monomeric and dimeric species has been noted. In fact, the paclitaxel dimer has also been observed under 95% water/5% acetonitrile conditions, which are much more like the in vivo conditions which would be found in patients. Indeed, the dimer may play a role in the bioactivity of paclitaxel (Balasubramanian et al.. 1994).

By extending this work to nanospray, it is expected to lower these detection limits significantly so that paclitaxel can be detected over the entire course of a 24-36 h blood plasma pharmacokinetics study. Thus, the nanospray emitters described here may be used to detect in vivo levels of paclitaxel and its metabolites. This is especially important because, to date, no determination has been achieved on the presence of in vivo levels of the paclitaxel dimer. which may play a significant role in the polymerization of tubuiin proteins to form microtubules. In addition, since sodiated paclitaxel dimer is the dominant dimeric species, nanospray can be used to examine whether high (mM) levels of sodium salts further enhance dimerization. Such studies would be virtually impossible with ESI since high salt concentrations make ESI virtually unusable: however, nanospray is much more tolerant of such high salt concentrations. LIST OF REFERENCES CITED

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Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

What Is Claimed:

1 A nanospray emitter comprising an emitter body comprising a fluid mlet. an outlet orifice, and a passage communicating between the fluid mlet and outlet orifice, and an electrically conductiv e polymer coating on at least a portion of the emitter body

2 The nanospray emitter according to claim 1. wherein the electrically conductiv e poly mer coating is a polyaniline coating

3 The nanospray emitter according to claim 1. wherein the emitter body comprises a glass or glass ceramic material

4 The nanospray emitter according to claim 3. wherein the glass or glass ceramic material is a borosilicate. aluminosilicate. or fused silica material

5 The nanospray emitter according to claim 1. wherein the emitter body is tapered toward the outlet orifice

6 The nanospray emitter according to claim 1 wherein the outlet orifice has an inner diameter of less than about 20 μm

7 The nanospray emitter according to claim 6. wherein the outlet orifice has an inner diameter of between about 1 μm to about 5 μm

8 The nanospray emitter according to claim 1. wherein the nanospray emitter is translucent

9 The nanospray emitter according to claim 1 , wherein the conductive polymer coating has a substantially uniform thickness of less than about 10 μm

10 The nanospray emitter according to claim 9, wherein the electrically conductive polymer coating has a substantially uniform thickness of between about 2 um to about 4 μm

1 1 The nanospray emitter according to claim 1. wherein the electrically conductive polymer coating comprises an electrical conductivity of between 1 0 10 to about 5 S/cm at 25°C

12 The nanospray emitter according to claim 1 , wherein the portion of the emitter body covered by the electrically conductive polymer is adjacent the outlet orifice ad|acent the inlet, or substantiallv the entire emitter body

13 The nanospray emitter according to claim 12. wherein the portion of the emitter body covered by the electrically conductive polymer is adjacent the inlet

14 The nanospray emitter according to claim 12. wherein the portion of the emitter body covered by the electrically conducti e polymer is adjacent the outlet orifice

15 A nano-electrospray device comprising a counter-electrode and a nanospray emitter according to claim 1 whose mlet is capable of fluid communication with a fluid source including a fluid containing one or more analytes. wherein either the counter-electrode or the nanospray emitter can be coupled to a power supply with the other being grounded, for development of a suitable electrical potential between the counter-electrode and the nanosprav emitter, which causes the fluid to be drawn through the passage of the nanosprav emitter for discharge from the outlet orifice

16. The nano-electrospray device according to claim 15. wherein the electrically conductive polymer coating is a polyaniline coating.

17. The nano-electrospray device according to claim 15. wherein the emitter body comprises a glass or glass ceramic material.

18. The nano-electrospray device according to claim 15. wherein the emitter body is tapered toward the outlet orifice.

19. The nano-electrospray device according to claim 15. wherein the outlet orifice has an inner diameter of less than about 20 μm.

20. The nano-electrospray device according to claim 15. wherein the nanospray emitter is translucent.

21. The nano-electrospray device according to claim 15. wherein the conductive polymer coating has a substantially uniform thickness of less than about 10 μm.

22. The nano-electrospray device according to claim 15. wherein the electrically conductive polymer coating comprises an electrical conductivity of between about 1.0 x 10^""' to about 5 S/cm at 25°C.

23. The nanospray emitter according to claim 15. wherein the portion of the emitter body covered by the electrically conductive polymer is adjacent the outlet orifice, adjacent the inlet, or substantially the entire emitter body.

24. A method for making a durable nanospray emitter, comprising: providing a nanospray emitter body comprising a fluid inlet, an outlet orifice, and a passage communicating between the fluid inlet and outlet orifice casting a thin film of an electrically conductive polymer coating onto at least a portion of the nanospray emitter body. 25 The method according to claim 24. wherein the conductive polymer coating is polyaniline

26 The method according to claim 24. wherein the emitter body comprises a glass oi glass ceramic

27 The method according to claim 24. wherein said casting comprises poly merizing the conductive poly mer onto the portion ot the emitter body in an acidic solution bv oxidation

28 The method according to claim 24. wherein said casting comprises coating the portion of the emitter body in a solution containing a non-conducti e base form of the conductive polymer: and doping the coated emitter to produce a conductive polymer coating

29 The method according to claim 24. wherein said casting comprises dipping the portion of the emitter body into a soluble suspension comprising the conducti e polymer

30 The method according to claim 29. wherein the soluble suspension comprises the conductive polymer in water

31 The method according to claim 29. wherein the soluble suspension comprises the conductiv e polymer in xvlene or a solvent comprising xy lene

32 The method according to claim 29. further comprising passing a fluid through the passage of the emitter body during said dipping 33 The method according to claim 32. wherein the fluid is a gas

34 The method according to claim 32. wherein the fluid is a non- reactiv e gas

35 The method according to claim 24. wherein said casting comππses introducing the emitter body , or portion thereof, into an RF discharge chamber and exposing the emitter bodv . or portion thereof, to aniline monomers under conditions effective to coating the portion of the emitter bodv with polv aniline

36 A method of forming a nanospray of a solution comprising passing a solution comprising an analyte through a nanospray emitter according to claim 1 under conditions effective to electrically charge the solution in a manner to emit the solution from the emitter in the form of a nanospray

37 A method of analyzing a solution for an analyte in the solution, the method comprising passing a solution comprising an analyte through a nanospray emitter according to claim 1 under conditions effective to electrically charge the solution in a manner to emit the solution from the emitter in the form of a nanospray. and analy zing the nanospray emission in a manner suitable to detect an anah te present in the solution

38 The method according to claim 37. wherein the analyte is a drug an endogenous biochemical, or a drug-protein complex