author(s) raoul kopelman, michael t. miller, murphy brasuel, …... · 2018. 2. 17. · modified...

Author(s)Raoul Kopelman, Michael T. Miller, Murphy Brasuel, Heather Clark, Marion Hoyer, and Martin Philbert

This article is available at IRis: http://iris.lib.neu.edu/bouve_fac_pubs/6

http://iris.lib.neu.edu/bouve_fac_pubs/6

Optochemical nanosensors for intracellular chemical measurement

Raoul Kopelmana, Michael T. Miller", Murphy Brasuela, Heather A. Clarka, Marion Hoyer",and Martin Philbert"

aDepartment of Chemistry and bDepartment of Environmental and Industrial Health,University of Michigan, Ann Arbor, MI 48 109-1055, USA

ABSTRACT

The development of a submicron optical fiber "supertip" has provided advantages over previously produced submicron tips,such as facilitating insertion of these sensors into cells while minimizing damage to the cell membrane. Fiber optic ioncorrelation-based nanosensors for sodium, potassium and chloride employing these "supertips" have been applied to then1nitoring of ion concentrations in single mouse oocytes. These sensors have also been used to monitor the effect of an ionchannel-blocking agent. In order to address the challenge associated with single-cell simultaneous measurement of multipleanalytes, the use of submicron optical fiber multiprobes has been explored.

Keywords: optodes, chemical sensors, fiber optic, multiprobe, intracellular measurements

1. INTRODUCTION

Single cell measurements can provide crucial information about biological processes and biochemical distributions thatcannot be obtained from bulk tissue Submicrometer sensors25 provide one method for real-time intracellularmeasurements. To be effective, these sensors must be highly selective, have fast response times, and cause minimal cellulardamage.

Submicrometer fiber optic sensors were first developed58using pulled optical fibers, similar to those utilized for near-fieldscanning optical microscopy,9 and a polyacrylamide matrix. Use of these submicrometer fibers was then extended to usewith poly(vinyl chloride), allowing decreased effects of photobleaching10 and detection of a greater number of

Here we present improved submicrometer fiber optic sensors and their application to the detection of various intracellularanalytes. The fiber tip shape has been redesigned to be less invasive and easier to insert into a cell. With this new "supertip", potassium, sodium, and chloride have been measured in viable single cells. In addition, a submicron multiprobe hasbeen developed for simultaneous detection of multiple analytes.

2. EXPERIMENTAL

1. Reagents

Poly(vinyl chloride) (PVC), potassium tetrakis [3,5-bis(trifluoromethyl)phenyllborate (KTFPB), chromoionophore I (ETH5294), chromoionophore II (ETH 2439), chromoionophore III (ETh 5350), potassium ionophore III (BME-44), bis(2-ethylhexyl)sebacate (DOS), and 2-nitrophenyloctyl ether (o-NPOE) were all obtained from Fluka. Tetrahydrofuran (THF),acrylamide, N,N-methylenebis[acrylamide], and triethylamine (TEA) were received from Aldrich. Kainic acid, M2 mediumand M16 medium and Sigmacote were obtained from Sigma. 1,1 '-dioctadecyl-3,3,3' ,3'-tetramethylindocarbocyanineperchlorate (Dii), Calcium Green- 1 hexapotassium salt, and Texas Red sulfonyl chloride were received from MolecularP. bes (Eugene, OR). Indium(III) octaethylporphyrin chloride, (In(OEP)Cl), was obtained from Midcentury (Posen, IL).The procedure described by Yamamoto and coworkers'2 was used to synthesize the 1,3-bridged calix[4]crown sodiumionophore.

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2. Optode film preparation

All film components were dissolved in freshly distilled THF. The exact film compositions are as follows: The potassium-sensitive film contained 3.2 mg of BME-44, 1 .5 mg of KTFPB, 0.9 mg of ETH 5350, 70.4 mg of DOS, and 34.6 mg ofPVC.The sodium-sensitive film contained 2.9 mg of 1,3-bridged calix[4Jcrown, 1.9 mg of KTFPB, 1.1 mg of ETH 5294, 76.1 mgof DOS, and 38. 1 mg of PVC. The chloride-sensitive film contained 2.8 mg of In(OEP)C1, I .8 mg of KTFPB, 1 .5 mg ofETH 2439, 1 30.4 mg of o-NPOE, 62. 1 mg of PVC, and 28 jiL of 1 .4 mM Dii solution.

3. Optodes

Liquid polymer-based sensors were fabricated with all-silica, single-mode optical fiber with a 3 xm core and an 80 jimcladding diameter (Thor Labs, Newton, NJ). Fibers were pulled to submicron size and aluminum-coated according to amodified procedure for preparation of near-field scanning microscopy light sources.9 The optodes were prepared by dippingthe pulled end of the optical fiber repeatedly (—5 times) into the membrane cocktail solution. The film thickness for optodesprepared in this manner is typically less than 300 nm.10

4. In vitro application

Mouse oocytes were obtained from the Transgenic Core Facility at the University of Michigan, where they were harvestedfrom superovulated C57BL/6 mice. They were maintained in M16 medium in a 5% C02, 37°C NuAire incubator (Plymouth,MN). For sensor studies, a single oocyte was transferred by micropipette to a 200 jiL drop of M2 medium on a Sigmacote-treated Petri dish. The dish was then filled with mineral oil and placed on the stage of an inverted microscope (see below)mounted on a vibration-isolated optical table. A micropipette manipulated by a three-axis positioner (see below) was used toimmobilize the oocyte over the microscope objective. The distal end of a liquid polymer-based optode was attached to amicromanipulator (Newport Model 150) so that it could be inserted into the oocyte in a precise manner. The proximal end ofthe optode was coupled into an argon ion laser (see below) exciting at 5 14.5 nm.

5. Photopolymerization somuons

Two distinct photopolymerization solutions were prepared. Solution 1 contained 0.04 mM Calcium Green-i, 36%acrylamide, and 4% N,N-niethylenebis[acrylamide] in 0.1 M phosphate buffer, pH 6.5. Solution 2 contained 0.3 mM TexasRed, 36% acrylamide, and 4% N,N-methylenebis[acrylamide] in 0.1 M phosphate buffer, pH 6.5.

6. Multiprobes

Submicron optical fiber multiprobes were provided by Physical Optics Corporation (Torrance, CA). They are comprised ofthree multimode optical fibers that have been bundled together at their distal ends and pulled down to a submicron tip. Thisdistal tip is silanized for 90 mm in a 2% solution of 3-(trimethoxysilyl)propyl methacrylate in pH 3.45 water and then dried inair. The silanized end, supported by a three-axis positioner (Newport 460A Series), was placed in a small Petri dishcontaining 500 jiL of polymerization solution and 20 jiL of TEA on the stage of an inverted microscope (see below). Theproximal ends of two fibers of a multiprobe were coupled into an argon ion laser (see below) exciting at 488 nm.Polymerization at spatially distinct regions on the multiprobe tip was controlled by allowing the laser beam to enter only thefiber of interest, while physically blocking the beam coupled to the other fiber. Polymer formation was monitored bycollecting fluorescence signal originating from the multiprobe tip. The fabricated sensors were dried and stored in air.

7. Experimental configuration

A schematic of the complete optical path and the sample stage has been previously reported.'3 The setup includes thefollowing: Ion Laser Technology (Salt Lake City, UT) argon ion laser and 488 nm or 5 14.5 nm laser band-pass filter(Newport, Irvine, CA); neutral density filters (Melles Griot); Uniblitz shutter controller (Rochester, NY); fiber coupler(Newport F-915 Series); Olympus inverted fluorescence microscope, IMT-II (Lake Success, NY); Nikon 50 mm f/1.8camera lenses; Acton 150 mm spectrograph (Acton, MA); and a Princeton Instruments 1024 x 256 LN2-cooled CCD array(Trenton, NJ). A beam-splitter (Melles Griot) and second fiber coupler were added to the setup for the multiprobeexperiments.

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1. Intracellular studies

3. RESULTS AND DISCUSSION

Several ion-selective fiber optic chemical sensors have been previously developed for use in intracellular applications.1''

The principle of operation of these liquid polymer-based sensors has been detailedelsewhere.'6 Previous reports have alsoinvestigated the dynamic range. selectivity and response time of each of these sensors. A recent modification of thesesensors that has been found to be beneficial to intracellular applications is a change inthe shape of the nano-optodes pulledfiber tip (see Figure 1). The previous tip shape tended to damage the cell upon insertion of the sensor, as it requiredthat a

relatively large hole be made in the cell membrane. The new "supertip' shape has a longer taper, which allows the sensors

insertion to produce . smaller hole in the membrane. In the present report. there are two intracellular studies presented. inthe first, sensors selective for potassium, sodium, and chloride have been applied to intracellular ion measurements in a singlemouse oocyte. The second monitors the affect of an ion channel-blocking agent on changes in intracellular ion

concentrations.

In the first study, measurements were made with each of the three distinct sensors, first in the culture medium, then inside thecytoplasmic space of the mouse oocyte. The data from these measurements are shown in Figures 2 and 3. The response ofion-correlation optodes is a function of the activity of the analyte ion and pH. For cation selective sensors, the response is afunction of the ratio of the analyte cation activity to the hydrogen ion activity (i.e. K'/H). For anion selective sensors, thisresponse is governed by the product of these activities (i.e. ClH). Intracellular levels of sodium are reported to be lowerthan those in the medium while intracellular potassium and chloride levels are reportedly higher.'7 Nanosensormeasurements indicated a decrease in the NafW when the sensor was inserted into the oocyte and an increase in theK/Hand the CFH. The system was then perturbed using a stimulant, kainic acid, which is known to open ion channels anddepolarize the cell. Indeed, a decrease in the K1H was seen upon addition of kainic acid. An increase in the NafH and theCl H were also observed in the presence of kainic acid. Even though each ion measurement is dependent on pH, when thedata were compared to one another, they did not follow a trend indicative of a simple pH change.

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Figure 1. Previous optical fiber tip shape (left) as compared to the new "supertip" shape (right).


Figure 2. Measures of potassium and sodium based on respective ion-sensitive optodes inserted into the cytoplasm ofsingle mouse oocytes. Values shown are proportional to ion activities. Measurements were taken in solution, in thecell, in the cell in the presence of kainic acid, and back in solution, respectively.

Outside Oocyte Inside Oocyte In Presence of Removed fromKainnic Acid Oocyte

Figure 3. Log of the product of the chloride and hydrogen ion activities (log([Cl]'[HJ)) from chloride-sensitiveoptode inserted into the cytoplasm of a single mouse oocyte. Measurements were taken in solution, in the cell, in thectil in the presence of kainic acid, and back in solution, respectively.

In the second set of experiments, potassium channels of some mouse oocytes were selectively blocked using TEA.Potassium fiber sensors were inserted into mouse oocytes treated with TEA or into untreated oocytes. and the cells were thenstimulated using kainic acid in the presence of sodium chloride, calcium chloride or potassium chloride. The influx of ionsfrom the extracellular space results in rapid depolarization of the membranes of excitable cells. Figure 4 shows intracellularresponses to sodium chloride, calcium chloride and potassium chloride monitored by the potassium fiber optode in thecytoplasm of a mouse oocyte in the absence of potassium channel blockers. The changes in K/H following addition ofsodium chloride, calcium chloride and potassium chloride are real-time measurements of ion flux following depolarization ofthe cell. The large signal change in response to low levels of K with respect to smaller signal changes induced by highlevels of Na and Ca2 indicates that the potassium nano-optodes were highly selective for potassium at physiologicallyrelevant concentrations of sodium and calcium. In the future, relationships between K and H need to he determined.

• K+ I H+

Outside Oocyte Inside Oocyte in Presence of Kainnic Removed from OocyteAcid

-6.2

-63

-64

C)

-66

-66

-6 7

-68

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202

1

0.7

0.6

Figure 4. Measurement of potassium/hydrogen in the cytoplasm of a mouse oocyte without a potassium-channelblocker (TEA). Values represent the ratiometric measurement of ion activities in the cell culture medium, inside theoocyte, then inside the stimulated oocyte in the presence of NaC1, CaC12, and KC1, respectively.

Figure 5 shows intracellular responses monitored by the potassium sensor in the presence of TEA. Note that depolarizationof the cell and the influx of sodium and calcium ions did not yield a change in the response of the potassium sensor. Inaddition, treatment of the cell with potassium chloride did not produce the intracellular response observed in the absence ofTEA. The lack of response after treatment with potassium chloride confirms cell viability and successful blockage ofpotassium channels in the oocyte.

:

I

0.9

0.8

0.7

0.6

0.5

Figure 5. Measurement of potassium/hydrogen in the cytoplasm of a mouse oocyte treated with 6 mM TEA. Valuesrepresent the ratiometric measurement of ion activities in the cell culture medium, inside the oocyte, then inside thestimulated oocyte in the presence of NaCl, CaCl2, and KC1, respectively.

0.9

0.8

0.5

Medium Oocyte 125 mM 0.2 mM 20 mM KCINaC1 CaCI2

Oocyte 10mM 125mM 0.2mM 20mMKainic NaC1 CaC12 KC1Acid


In summary. we have observed three distinct analytes with fiber optic probes, inserted one at a time. It is straightforwardtogeneralize this to many additional analytes, such as nitrite and pH. However, it gets increasingly challenging to insert morethan two fiber sensors at a time into an oocyte (see Figure 6). A better solution presents itself through the use of optical fibermultiprohe sensors.

Figure 6. Two fiber optic nano-optodes inserted into a single mouse oocyte.

2. Multiprobe experiments

In an effort to work toward simultaneous multianalyte measurement, preliminary investigations have begun employingoptical fiber multiprobes. The design of these multiprobes allows each fiber to deliver light signal to a correspondinglocation at the multiprobe's submicron tip (see Figure 7). Photopolymerization at different tip locations was accomplished bysequentially coupling different fibers of one multiprobe to an argon ion laser. Dyes having different emission spectra wereentrapped within the polymer at different tip locations in order to determine the extent of cross-talk in the completed sensor.Early data reveals only minimal cross-talk between two fibers of a three fiber multiprobe (see Figure 8). However, furtherstudies have suggested wicking (via capillary action) of the dye-containing photopolymerization solution into cavities ofsome multiprobes. which could contribute to the delocalized fluorescence signal that has been observed. These problemsmust be addressed so that fluorescence signal from various ion-sensitive dyes can he selectively excited. This selectiveexcitation is prerequisite for intracellular multianalyte detection to be possible.

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250

00C

.E 150a)(.)C0)0ci 1000)0

50

0

500 750

Wavelength (nm)Figure 8. Multiprobe sensor with sensors polymerized through two distinct fibers of the three fiber bundle. Fiber 1corresponds to the region of the multiprobe tip where Calcium Green-I was entrapped, while Fiber 2 corresponds tothe region containing Texas Red. The two regions are <500 nm apart.

204

Figure 7. SEM of optical fiber multiprobe submicron tip. Scale bar represents 1 m.

550 600 650 700


4. ACKNOWLEDGEMENTS

We thank the Transgenic Core Facility of the University of Michigan for providing the mouse oocytes. We also thankPhysical Optics Corporation for providing us with the optical fiber multiprobes. We acknowledge DARPA grant MDA 972-97-0006 and NIH grant ROl GM50300 for financial support.

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author(s) raoul kopelman, michael t. miller, murphy brasuel, …... · 2018. 2. 17. · modified...

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