Electrospun Fibers as a Solid-State Real-Time Zinc Ion Sensor with High Sensitivity and Cell Medium Compatibility

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Jia-Hao Syu , Yi-Kai Cheng , Wun-Yan Hong , Hsing-Ping Wang , Yu-Chao Lin ,

Hsin-Fei Meng , * Hsiao-Wen Zan , Sheng-Fu Horng , Gao-Fong Chang ,

Chen-Hsiung Hung , * Yu-Cheng Chiu , Wen-Chang Chen , * May-Jywan Tsai ,

and Henrich Cheng

1. Introduction

Solid-state real-time chemical sensors are important for biomed-ical research and medicine. Conventionally, the target chemi-cals are detected by dissolving the probe molecules in liquid,

then the color or fl uorescence changes result from the reaction with the target. The probe molecules may also interfere with the cell or tissue that releases the chemicals. In other words such a soluble probe is invasive. On the other hand a solid-state sensor is non-invasive and it transforms the real-time chemical con-centration into electronic signals without interference with the biological processes under study. Zinc II ions play several key roles in animals and their pathology. Zinc ions play key roles in normal physiology and in pathological conditions. [ 1–3 ] _{It is a}

transynaptic mediator. Most Zn + 2 _{in the}

brain is tightly bound or sequestered in cellular compartments. High levels of zinc release in the synapse contribute to the selective nerve cell injury from stroke and from Alzheimer’s disease. [ 4 , 5 ] _{Zinc is the}

second most abundant transition metal ion in the human body after iron. Around the neural axon the zinc ion concentration is as high as 10 − 6 _{M . In order to study the dynamics of biological}

processes such as stroke, it is important to detect the zinc ion concentration every few minutes. Such real-time detection is

Electrospun Fibers as a Solid-State Real-Time Zinc

Ion Sensor with High Sensitivity and Cell Medium

Compatibility

meso -2,6-Dichlorophenyltripyrrinone (TPN-Cl 2 ), a probe molecule for zinc II ions, is dispersed in a polymer host. The red fl uorescence peak at 620 nm appears when the molecule forms a complex with zinc at its center. TPN-Cl 2 has a high selectivity for zinc II and tolerates many common metal ions present in the human body. The probe molecules are blended with a hydrogel polymer, poly(2-hydroxyethyl methacrylate) (poly HEMA), with 30 wt% dimethylformamide (DMF). The fi ber structure with 1 μ m diameter is made by electrospinning in DMF solution of the probe and poly HEMA mixture. The fi brous fi lm detects zinc ions with concentrations as low as 10 − 6 _{M in} real-time both in water and in the commonly used cell culture liquid media Dulbecco’s modifi ed Eagle medium (DMEM) and fetal bovine serum (FBS), which contain many metal ions and proteins. The time-resolution is 5 min for 10 − 6 _{M and 1 min for 10} − 5 _{M. This sensitivity and response speed satisfy the} requirements for non-invasive biomedical studies.

DOI: 10.1002/adfm.201201242

J.-H. Syu, Y.-K. Cheng, W.-Y. Hong, Y.-C. Lin, Prof. S.-F. Horng

Department of Electrical Engineering National Tsing Hua University Hsinchu 300, Taiwan, Republic of China H.-P. Wang, Prof. H.-F. Meng

Institute of Physics

National Chiao Tung University Hsinchu 300, Taiwan, Republic of China E-mail: meng@mail.nctu.edu.tw Prof. H.-W. Zan

Department of Photonics and the Institute of Electro-Optical Engineering

National Chiao Tung University Hsinchu 300, Taiwan, Republic of China

G.-F. Chang, Prof. C.-H. Hung Institute of Chemistry

Academia Sinica, Taipei 115, Taiwan, Republic of China E-mail: chhung@chem.sinica.edu.tw

Y.-C. Chiu, Prof. W.-C. Chen Department of Chemical Engineering National Taiwan University

Taipei 106, Taiwan, Republic of China E-mail: chenwc@ntu.edu.tw M.-J. Tsai, Prof. H. Cheng

Neural Regeneration Laboratory and Center for Neural Regeneration Department of Neurosurgery

Neurological Institute

Taipei Veterans General Hospital Taipei 112, Taiwan, Republic of China

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porous host with similar fl uorescence response to the zinc ions in water and in the liquid buffer medium used for cell culture. The contents of two typical liquid media, Hank’s balanced salt solution (HBSS) and DMEM, are listed in Table 1 . DMEM con-tains many proteins and more ions than HBSS, and it is com-monly used for living cells in cultures. For example, Fe III is present in DMEM but not in HBSS. Our goal is therefore to develop a sensor that can function in DMEM, which is more challenging than HBSS.

TPN-Cl 2 molecules are blended with poly HEMA host for

fi ber and planar structures for comparison. For the planar structure, the monomers are mixed with the probe followed by a thermal curing to transform the mixture to a matrix of poly HEMA. [ 9 ] _{The resulting planar fi lm is shown in Figure 3 C.}

Electrospinning system shown in Figure 3 A is used to gen-erate the fi ber structure. [ 11–13 ] _{Under the high voltage between}

the nozzle and the substrate, the solution is charged and fi ne fi bers form due to Coulomb repulsion. [ 14 ] _{In order to provide}

enough viscosity for the electrospinning poly HEMA rather than the monomers are mixed with TPN-Cl 2 in DMF

solu-tion at 30 wt% with a molecular weight of 300 000 (300 k) for poly HEMA, and TPN-Cl 2 in DMF solution at 2.52 × 10 − 2 wt%.

The resulting fi ber fi lm is shown in the scanning electron impossible for conventional molecular probes dissolved in

solu-tion. In practice, the solid-state zinc sensor must function not in water but instead in liquid biological medium, which usually contains many other ions and proteins. Those contents of the medium should not interfere with the detection and the sensor structure should be stable for long immersion times in the medium. To date most zinc ion detection is based on soluble fl uorescent molecular probes [ 6 , 7 ] _{or dispersed nanoparticles.} [ 8 ]

Despite their high sensitivity they are invasive and do not give real-time results. Recently we reported a solid-state zinc sensor with a fl uorescent molecule in a polymer host. [ 9 ] _{The sensitivity}

of 10 − 4 _{M is, however, not enough for biomedical applications}

and the detection was measured in water rather than the com-plex cell buffer medium. A highly sensitive solid-state zinc sensor that is stable in the common cell buffer medium has not been reported.

Here, we developed a solid-state zinc ion sensor with electro-spinning fi bers containing a highly selective fl uorescent probe molecule TPN-Cl 2 . [ 10 ] The TPN-Cl 2 probe has red fl uorescence

only when it captures a zinc ion at its center. The molecules are blended with a host polymer, poly(2-hydroxyethyl methacrylate) (poly HEMA), and do not diffuse into the liquid medium after long immersion times. The polymer host containing the probe is made in fi ber form using electrospinning with a fi ber dia-meter around 1 μ m. Because of the large surface area of the fi ber fi lm the zinc ion sensitivity is enhanced from 10 − 4 _{M for a planar}

fi lm to 10 − 6 _{M for a fi ber fi lm. Such a concentration occurs near}

the neural axon region and is a criterion for the biological appli-cations of the developed sensor. The TPN-Cl 2 probe is able to

detect zinc ions with high tolerance to the presence of a wide range of metal ions. As a result, the sensor works well in both water and the complex cell buffer medium that contains many other ions. The fi ber fi lm is also highly stable in acid. The time resolution is about 1 min for a typical zinc concentration of 10 − 5 _{M . This rapid time response makes the sensor a possible}

tool to monitor rapidly changing processes, such as stroke, which have time scales of hours.

2. The Probe Molecule and Fiber Film

The zinc ion probe TPN-Cl 2 is shown in Figure 1 . When a

zinc ion is captured it becomes a complex, TPN-Cl 2 -Zn, which

is shown in Figure 1 . The photoluminescence (PL) spectrum under excitation at 565 nm wavelength in methanol solution before and after Zn chelating is shown in Figure 2 A. The red fl uorescence peaked at 620 nm is present only in TPN-Cl 2 -Zn.

The turn-on of PL can be observed for Zn ion concentrations as low as 10 − 8 _{M in methanol solutions. The PL is not turned on by}

other common ions, as shown in Figure 2 B, where the concen-tration of other metal ions is 2 × 10 − 5 _{M and the concentration of}

TPN-Cl 2 is 10 − 6 M . Furthermore, the response to Zn ions tolerates

the presence of most common ions; this is shown in Figure 2 B, where metal ions are added before zinc. In particular, Mg II is tolerated in contrast to the probe m -benziporpho dimethene (BPDM-H), which we studied previously (Figure 2 C). [ 9 ] _Mn

II, Co II, and Ni II do interfere with zinc detection, but their concentrations in animals are very low. In an ideal solid-state sensor structure the zinc probe molecules are stably fi xed in a

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microscopy (SEM) images in Figure 4 A. The fi ber diameter is about 1 μ m. There is some swelling of the fi ber after water immersion, as shown in Figure 4 B. The water permeability is expected to be reduced by the swelling. High molecular weight of poly HEMA is shown to be important to reduce the

Table 1. The content of a two typical liquid media: DMEM and HBSS.

DMEM Components [m M ] Amino Acids Glycine 0.4 L -Arginine hydrochloride 0.398 L -Cystine 2HCl 0.201 L -Histidine hydrochloride-H 2 O 0.2 L -Isoleucine 0.802 L -Leucine 0.802 L -Lysine hydrochloride 0.798 L -Methionine 0.201 L -Phenylalanine 0.4 L -Serine 0.4 L -Threonine 0.798 L -Tryptophan 0.0784

L -Tyrosine disodium salt dihydrate 0.398

L -Valine 0.803 Vitamins Choline chloride 0.0286 D -Calcium pantothenate 0.00839 Folic Acid 0.00907 Niacinamide 0.0328 Pyridoxine hydrochloride 0.0196 Ribofl avin 0.00106 Thiamine hydrochloride 0.0119 i-Inositol 0.04 Inorganic Salts

Calcium chloride (CaCl 2 ) (anhyd.) 1.8 Ferric nitrate (Fe(NO 3 ) 3 -9H 2 O) 0.000248 Magnesium sulfate (MgSO 4 ) (anhyd.) 0.814

Potassium chloride (KCl) 5.33

Sodium bicarbonate (NaHCO 3 ) 44.05

Sodium chloride (NaCl) 110.34

Sodium phosphate monobasic (NaH 2 PO 4 -H 2 O) 0.906 Other Components

D -Glucose (Dextrose) 5.56

Sodium pyruvate 1

HBSS Components [m M ]

Inorganic Salts

Calcium chloride (CaCl 2 ) (anhyd.) 1.26 Magnesium chloride (MgCl 2 -6H 2 O) 0.493 Magnesium sulfate (MgSO 4 -7H 2 O) 0.407

Potassium chloride (KCl) 5.33

Potassium phosphate monobasic (KH 2 PO 4 ) 0.441 Sodium bicarbonate (NaHCO 3 ) 4.17

Sodium chloride (NaCl) 137.93

Sodium phosphate dibasic (Na 2 HPO 4 ) anhydrous

0.338

Other Components

D -Glucose (Dextrose) 5.56

Figure 2 . A) The photoluminescence (PL) spectrum of TPN-Cl 2 in

meth-anol solution before and after Zn chelation. B) TPN-Cl 2 ’s specifi city: the

concentration of TPN-Cl 2 is 10 − 6 M and the concentration of all other metal

ion is 2 × 10 − 5 _{M . C) BPDM-H’s specifi city: the concentration of BPDM-H}

is 3 × 10 − 6

M and the concentration of all other metal ion is 6 × 10 − 5 M .

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swelling, as shown in Figure 4 C,D. The fi ber fi lm on the sub-strate is shown in Figure 3 B with a thickness around 0.87 mm. The PL spectra for the fi ber and planar fi lm in water, with or without zinc ions, are shown in Figure 5 A–E and Figure 6 A–C. The PL spectra for TPN-Cl 2 in solution and in the fi ber are

similar, with the main peak at 620 nm being responsive to the

zinc ions. Before the introduction of the zinc ions, there is a clear PL background in the fi ber but the background is barely detectable in solution. The fi ber network may inhibit a vibra-tion mode, which would quench the 620 nm emission of free TPN-Cl 2 in solution. The similarity of the PL spectra in

solu-tion and fi ber suggests that there is no chemical reacsolu-tion and the physical interactions between the fi ber host and the TPN-Cl 2 probe molecules are rather weak. On the other hand the

main peak of the PL spectrum of the planar fi lm is changed to 570 nm, which does not respond to zinc ions. The 620 nm emission becomes a shoulder in the spectrum and remains responsive to zinc ions. The difference in the PL spectra in the planar structure relative to the fi lm and to solution and fi ber implies that the physical interaction is stronger and the geometry of the probe molecules is distorted. For comparison, the PL of the previous probe, BPDM-H, with zinc ions in the planar fi lm is also shown.

3. Sensor Results and Discussions

A microfl uidics system is shown in Figure 7 , where a poly-dimethylsiloxane (PDMS) mold on glass is used to rapidly change the zinc ion concentration. [ 9 ] _{A syringe is used to}

inject solution with zero or various zinc concentration into the channel. The sensing fi lm is immersed in the liquid inside the channel. The fi lm is constantly excited by 405 nm laser

Figure 3 . A) The electrospinning system. B) The fi ber fi lm on the

sub-strate. C) The planar fi lm.

Figure 4 . The SEM image of the fi ber fi lm. A) 300 k poly HEMA before water immersion. B) 300 k poly HEMA after water immersion. C) 1 M (1 million)

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in the selectivity shown in Figure 2 C above. The result for TPN-Cl 2 planar fi lm is shown in Figure 8 C. It turns out that

such a fi lm works well in pure DMEM. In standard cell cul-ture DMEM is mixed with the fetal bovine serum (FBS) with a volume ratio 9:1. As such, FBS is added to the poly HEMA fi lm containing TPN-Cl 2 and it still responds well to Zn II ions, as

shown in Figure 8 E. The functioning in DMEM plus FBS is a highly desirable property. The compatibility of TPN-Cl 2 fi lm in

DMEM demonstrates again that this probe is highly selective to zinc II and tolerates the presence of a wide range of chemi-cals including organic molecules and metal ions. The fi lm has an almost exclusive response to zinc ions, even in a complex environment. The slope of the fl uorescence as a function of time can be used to decide the real-time zinc ion concentra-tion. The time resolution is defi ned as the interval required to diode with pulse width of 10 s and period of 60 s. The PL is

registered by a charge-coupled-device (CCD) camera. The PL signals at the peak wavelength of 620 nm are shown in Figure 8 C–E as a function of time for the TPN-Cl 2 sensing

fi lm under different cell culture liquid media. Even though this work is focused on the probe TPN-Cl 2 , the results of the

previous probe BPDM-H in the same setup are presented as a comparison in order to illustrate their difference in toler-ance to the culture media. The former turns out to have a far better tolerance than the latter, as shown. Consider BPDM-H fi rst, Figure 8 A is the PL spectrum of BPDM-H planar fi lm, and Figure 8 B shows that while it works well in HBSS, no response to zinc ion is observed when more than 10 vol% of DMEM is added. Presumably the Mg II in DMEM occupies the center of BPDM-H and block the zinc ions as suggested

Figure 5 . Time dependence of the PL spectra for the fi ber fi lm in deionized water (DI water) with zinc ions, measured with a PL spectrometer:

A) 0 M zinc ions; B) 10 − 6 M zinc ions; C) 10 − 5 M zinc ions; and D) 10 − 4 M zinc ion. E) The PL spectra in DI water with different concentrations of zinc ions.

550 600 650 700 750 0 50 100 150 A) B) C) D) Zn+2: 0M

Fiber film in DI water

Phot olu minesc ence (a .u.) Wavelength (nm) 0 min 5 min 10 min 15 min 550 600 650 700 750 0 50 100 150 Phot oluminesce nce (a.u) Wavelength(nm) 0 min 5 min 10 min 15 min 20 min Zn+2:10-6M

Fiber film in DI water

550 600 650 700 750 0 100 200 300 400 500 600 700 Phot olu minesc ence (a .u.) Wavelength (nm) 0 min 2 min 4 min 6 min 8 min 10 min Zn+2:10-4M

Fiber film in DI water

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Fiber film in DI water

E) 0 20 40 60 80 100 100 200 300 400 500 600 700 10-4M 10-5M 0M 0M Pho tolum in esc e nc e ( a .u.) Time (minute)

TPN-Cl₂ fiber film in DI water

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identify a clear difference in the slope. For TPN-Cl 2 the time

resolution is about 5 min for a zinc concentration of 10 − 4 _{M ,}

both in water and pure DMEM. It can be further improved by the fi ber fi lm as described below.

So far the zinc ion detection has been done in a planar fi lm. The detection limit is 10 − 4 _{M , as shown in Figure 6 A−C. The}

detection limit of the fi ber fi lm for TPN-Cl 2 in poly HEMA

host is presented in Figure 5 A–E. Despite of the less swelling of the 1 million molecular weight poly HEMA shown in Figure 4 B,D, no signifi cant improvement in the sensitivity is observed as the molecular weight is increased from 300 k to 1 million. Due the larger fi ber fi lm deposition area and easier control during the electrospinning process, the 300 k poly HEMA is used in all the fi ber data below unless otherwise specifi ed. For better spectral resolution, instead of using the CCD camera, the spectra are recorded using a PL spectrometer and the fi lm is immersed in a quartz vessel. The time depend-ence of Figure 5 E is shown by plotting the PL value at 620 nm (recorded using a spectrometer) versus time, under changing zinc concentration in the quartz vessel. For water solution the zinc ion concentration is changed from 10 − 6 _{M to 10} − 4 _{M .}

The fi ber fi lm has a clear response to zinc ions for concentra-tions as low as 10 − 6 _{M . The sensitivity is therefore improved by}

two orders of magnitude as the fi lm is changed from planar to fi brous with much larger surface area. Similar detection limit of 10 − 6 _{M is obtained from TPN-Cl}

2 fi ber fi lm in pure

DMEM solution as shown in Figure 9 A–E. In Figure 9 A the PL at peak wavelength of 620 nm is plotted as a function of time for various zinc ion concentrations for the fi ber fi lm. Different concentrations gives different slope values, which are used to measure the sensing. For zinc ion concentrations of 10 − 6 _M

the resolution is about 5 min, whereas the time resolution is raised to 1 min for a concentration of 10 − 5 _{M . As expected there}

is a trade-off between the concentration and the response time. Nevertheless 5 min resolution at concentrations as low as 10 − 6 _M

is expected to satisfy the requirements for some biomedical applications. The PL responses of fi ber fi lms made of different poly HEMA molecular weights are compared. Even though the level of swelling is lower in high molecular weight fi ber as shown in Figure 4 B,D, the sensing properties are quite similar for the two molecular weights. All the original separated fi ber structures are destroyed by the immersion regardless of the molecular weight. In order to improve the fi lm sensitivity from 10 − 6 _{M to 10} − 8 _{M free probe in solution, the fi ber host with high}

stability in the solution may be necessary to allow for rapid zinc ion diffusion.

Figure 6 . Time dependence of the PL spectra of the planar fi lm in DI water with zinc ions measured with a PL spectrometer: A) 10 − 5 _{M zinc}

ions; B) 10 − 4 _{M zinc ions; and C) 10} − 3 _{M zinc ions.}

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Figure 7 . Microfl uid system. The sensing fi lm is the fi ber or planar fi lm

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the fi ber fi lm immersed in an acid aqueous solution of pH 3.31. A measure of the response of the fi ber fi lm to zinc ions is the differ-ence in the 620 nm PL at 30 min (when zinc ions are added) and at 35 min in Figure 10 . Such a response is plotted as a function of the pH value in Figure 10 E. It suddenly drops to almost zero as the pH changes from 3.31 to 2.38; the sensor therefore may not work well in very acid parts of the body such as the stomach.

4. Conclusion

In conclusion, a solid-state sensing fi lm is developed to detect zinc II ions within the physiological concentration range in complex liquid medium. The fl uorescent molecular probe embedded in the hydrogel polymer host responds exclusively to zinc ions, even when many other ions and proteins are present in the medium. The fi lm is made in fi ber form by an electros-pinning method in order to raise the sensitivity. In the medium Finally we show that the TPN-Cl 2 fi ber fi lm is stable and

remains responsive to zinc ion even in a very acid condition. The pH value of water is tuned by adding hydrochloric acid (HCl). The PL spectrum for various pH values is shown. For pH between 6.16 and 3.31 the PL response to zinc ion remains the similar and its intensity drops and shape changes only at pH of 2.38. The sensing fi lm therefore works well unless the condi-tions are very acid (pH below 3). In the PL responses shown in Figure 5 A–E above, the pH of the water solution changes slightly as zinc acetate, the source of zinc ions, is added. The pH versus time is shown in Figure 10 F. Because the pH is always above 5 in the safe range shown in Figure 10 A–D, the possibility that the PL modulation in Figure 5 A–E results from pH variation is ruled out. Even though most of the human body remains neutral, some local areas may be quite acid. It is therefore useful if the sensor can operate under acid conditions. Figure 10 C shows that the PL still respond well to zinc ion concentration of 10 − 3 _{M for}

Figure 8 . The PL at its peak wavelength as a function of time for the sensing fi lm under buffer. All data were measured with a CCD camera. A) The PL

spectrum of BPDM-H planar fi lm. B) The working conditions of the BPDM-H planar fi lm made by the buffer solutions with different ratio. The time-dependent PL is taken at 680 nm. C–E) The time-time-dependent PL of TPN-Cl 2 is taken at 620 nm. C) The planar fi lm under pure DMEM. D) The fi ber fi lm

under pure DMEM. E) The fi ber fi lm under DMEM + 10% FBS.

650 700 750 800 850 900 0 200 400 600 800 1000 A) C) B) D) Photoluminescence (a. u.) Wavelength (nm) BPDM-H planar film 0 5 10 15 20 25 30 400 600 800 1000 10-3M 0M Ph oto luminescen ce (a.u.) Time (minute) HBSS:DMEM = 100: 0 HBSS:DMEM = 95: 5 HBSS:DMEM = 90:10 0M BPDM-H planar film in buffer solutions 0 20 40 60 80 600 800 1000 1200 1400 1600 0M 0M 10-3M 10-4M 0M Pho tol u m in e scen ce ( a.u .) Time (minute)

TPN-Cl₂ palnar film in pure DMEM

0 20 40 60 80 100 120 600 800 1000 1200 1400 1600 ₁₀-4_{M 10}-3_{M 0M} 0M 0M

TPN-Cl₂ fiber film in pure DMEM

10-5M Pho tol u min escenc e ( a .u .) Time (minute) 0M E) 0 20 40 60 80 100 120 500 1000 1500

TPN-Cl₂ fiber film in DMEM+10% FBS

Pho tolumin escenc e ( a .u.) Time (minute) 10-3_M 10-4_{M 0M 0M} 0M 10-5M 0M

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028-MY3, Veterans General Hospitals and University System of Taiwan Joint Research Program(VGHUST100-G5-1-1), and the Academic Sinica under Contract No. AS-100-TP-B03. The authors are grateful to the members in the Biomimetic System Research Center at National Chiao Tung University.

Received: May 7, 2012 Revised: July 6, 2012 Published online: October 23, 2012

such a fi lm respond to zinc ions with concentrations as low as 10 − 6 _{M with a time resolution of 5 min. The fi ber fi lm shows}

good stability and unchanged sensing functions in high acidic conditions. Zinc ion plays crucial roles in brain function and immune system. The sensitivity and respond speed of the fi lm makes it possible for a non-invasive and real-time study for the variation of zinc ion concentration in important biological processes.

Acknowledgements

J.H.S. and H.F.M. thank Yu-Fan Chang for useful discussions. This work was supported by the Ministry of Economic Affairs of Taiwan under Contract No. 99-EC-17-A-07-S1-157, National Science Council of Taiwan under Contract No. 99-2628-M-009-001- and

No.98-2112-M-007-0 40 80 120 160 200 100 200 300 400 500 600 A) C) B) D)

TPN-Cl₂ fiber film in DMEM

10-4 M 10-5 M 10-6M 0M 0M Pho tol u m in e scen ce ( a.u .) Time (minute) 0M 550 600 650 700 750 0 20 40 60 80 100 120 TPN-Cl₂ fiber film Phot olu minesc ence (a .u.) Wavelength (nm) 0 min 5 min 10 min 15 min 20 min 25 min 30 min 300k poly HEMA Zn+2:10-6M 550 600 650 700 750 0 40 80 120 160 200 TPN-Cl₂ fiber film Zn+2:10-5M 300k poly HEMA Phot olu minesc ence (a .u.) Wavelength (nm) 0 min 5 min 10 min 15 min 20 min 550 600 650 700 750 10 20 30 40 50 60 70 TPN-Cl₂ fiber film Wavelength (nm) Pho tolum in escenc e ( a .u.) Zn+2:10-6M 1M poly HEMA 0 min 5 min 10 min 15 min 20 min 25 min E) 550 600 650 700 750 20 40 60 80 100 TPN-Cl 2 fiber film Wavelength (nm) Pho tolumin e scenc e ( a .u.) Zn+2:10-5M 1M poly HEMA 0 min 5 min 10 min 15 min

Figure 9 . A) The time-dependent PL spectrum taken at 620 nm. TPN-Cl 2 fi ber fi lm works well in pure DMEM solution. The time-dependent PL was

recorded at 620 nm, measured by a PL spectrometer. B–E) The response of the PL spectra to zinc ions, for fi ber fi lms of 300 k and 1 M poly HEMA molecular weight in DMEM solution.

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600 650 700 750 0 200 400 600 800 1000 A) C) Zn2+:10-3M Ph oto lum in escen ce (a. u. ) Wavelength (nm) 0min 5min 10min 10min 15min 20min 25min 30min 30min 35min 40min 45min 50min TPN-Cl

2 Fiber film DI water pH=6.16 B) 600 650 700 750 0 200 400 600 800 1000 1200 Fiber film Ph oto luminescen ce (a.u.) Wavelength (nm) 0min 5min 10min 10min 15min 20min 25min 30min 30min 35min 40min 45min 50min DI water pH=4.28 Zn2+:10-3M 600 650 700 750 0 100 200 300 400 500 Fiber film Ph oto luminescen ce (a.u. ) Wavelength (nm) 0 min 5 min 10 min 10 min 15 min 20 min 25 min 30 min 30 min 35 min 40 min 45 min 50 min DI water pH=3.31 Zn2+:10-3M D) 600 650 700 750 20 40 60 Fiber film Pho tolumin escenc e ( a .u ) Wavelength (nm) 0min 5min 10min 10min 15min 20min 25min 30min 30min 35min 40min 45min 50min DI water pH=2.38 Zn2+:10-3M E) 0 200 400 600 800 (35min PL) - (30min PL) PL di ff erence (a. u .) pH 6.16 4.28 3.31 2.38 F) 0 10 20 30 40 50 60 5.0 5.5 6.0 6.5 7.0 7.5 pH Time(minute) 10-3 M 10-4 M 10-5 M The pH of different Zinc ion

concentration in DI water

Figure 10 . The PL spectra at different times for various pH values in DI water: A) pH = 6.16; B) pH = 4.28; C) pH = 3.31; and D) pH = 2.38. E) The difference in PL signal at 620 nm at 35 min and 30 min as a measure of response to zinc ions for various pH values. F) The variation of the water pH value after addition of zinc acetate at three concentrations.

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