Molecularly Imprinted Membrane Electrospray Ionization for Direct

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Molecularly Imprinted Membrane Electrospray Ionization for Direct Sample Analyses Tianyi Li, Liusheng Fan, Yingfeng Wang, Xuebin Huang, Jianguo Xu, Jinxing Lu, Mei Zhang, and Wei Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02571 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016

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Analytical Chemistry

Molecularly Imprinted Membrane Electrospray Ionization for Direct Sample Analyses Tianyi Li1,5†, Liusheng Fan2†, Yingfeng Wang3, Xuebin Huang4, Jianguo Xu1,5, Jinxing Lu1,5, Mei Zhang1,5* and Wei Xu2*

1

State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing China, 102206; 2 School of Life Science, Beijing Institute of Technology, Beijing China, 100081; 3 Department of Chemistry, Capital Normal University, Beijing China, 100048; 4 School of Chemistry, Beijing Institute of Technology, Beijing China, 100081; 5 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou China, 310003. †

Equal contribution

*Co-corresponding Authors: Prof. Wei Xu School of Life Science, Beijing Institute of Technology Beijing China, 100081 Email: [email protected]; Phone: +86-10-68918123 Prof. Mei Zhang State Key Laboratory for Infectious Disease Prevention and Control Beijing China, 102206 Email: [email protected]; Phone: +86-10-58900749

ACS Paragon Plus Environment


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ABSTRACT Typically dealing with practical samples with very complex matrices, ambient ionization mass spectrometry suffers from low detection sensitivity. In this study, molecular imprinting technology was explored and integrated with membrane electrospray ionization (MESI) method for direct sample analyses. By enriching targeted analytes on molecularly imprinted membrane (MIM), 10- to 50-fold limit of quantitation

improvement

could

be

achieved

compared

to

conventional

nano-electrospray ionization method or other ambient ionization method. Molecularly imprinted membrane (MIM) was prepared by cross-linking synthesized molecularly imprinted polymer layer onto PVDF membrane. The characteristics of MIM in recognizing target analytes were investigated and verified. Experiments showed that MIM-ESI could provide satisfactory performances for direct quantification of targeted analytes in complex samples using MS, and the quantitative performance of this methodology was validated. With the capability of target enrichment, the uses of MIM-ESI MS in different application fields were also demonstrated, including food safety, quantification of drug concentrations in blood, pesticide residues in soil and antibiotic residues in milk.

Keywords: molecularly imprinted membrane; electrospray ionization; ambient ionization; mass spectrometry


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1. INTRODUCTION Mass spectrometry (MS) has been used in many fields with its advantages of high sensitivity, high resolution and small sample consumption. With the developments of ambient ionization technologies, direct sample analysis in an open environment is allowed for MS analysis. Techniques, such as direct analysis in real-time (DART),1 electrospray ionization (DESI),2 paper spray ionization (PS)3 etc.,4-23 could achieve direct analyte detections in complex matrices with minimized sample handling process. Previously, membrane electrospray ionization (MESI) was developed in our group,24 in which membrane was applied for three-dimensional molecule separation in real-time. Despite MESI is able to remove matrices during the analysis process, the capability of quantifying a target analyte with low LOQ and wide dynamic range is still challenging. Many molecule enrichment methods have been reported and coupled with ambient ionization. For example, micro-extraction permitted sample preparation directly coupled with MS ionization source.25-29 Furthermore, molecular imprinting technique is another useful strategy for target analyte enrichment, which was first proposed by Linus Pauling.30 And then it has been continuously corrected by many chemists, and ultimately led to a novel field of polymer imprinting.31-35 Owing to its advantages of structural predetermination, specific recognition and practicality, molecular imprinting technology has been widely used in many fields,



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including chiral separation,36 medicine analysis,37 solid phase extraction,38 biomimetic sensor,39 enzyme-catalyzed synthesis,40 and so on.41,42 In this study, molecular imprinting technology was coupled with MESI to achieve low concentration of targeted analytes. Synthesized molecular imprinting polymer (MIP) was cross-linked onto PVDF membrane to achieve target molecule enrichment. Then this membrane could be used for direct ionization and mass analysis. After optimization, MIM-ESI was able to achieve 10- to 50-fold sensitivity improvement compared with the nanoESI or other ambient ionization method. MIM-ESI has also been demonstrated for multiple applications, including quantifications of additive in urine, drug concentrations in blood, pesticide in soil and antibiotic residues in milk samples.

2. EXPERIMENTAL SECTION Chemicals and materials Hydrophilic and hydrophobic polyvinylidene difluoride (PVDF) membrane, hydrophilic polytetrafluoroethylene (PTFE) membrane and hydrophilic regenerated cellulose (RC) membrane were purchased from Agela Technologies (Tianjin, China; Φ 25 mm, 0.45 µm pore size); ractopamine hydrochloride (RAC), clenbuterol hydrochloride (CLE), trimetoprim (TMP), methotrexate (MTX), tolclofos-methyl (TCFM),

chlorpyrifos

2,2'-azoisobutyronitrile

(CPF),

norfloxacin

(AIBN),

ethylene

(NFLX), glycol

ciprofloxacin

dimethacrylate

(CPFX),

(EGDMA),

methacrylic acid (MAA), acrylic acid (AA) and potassium peroxydisulfate (KPS)


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were purchased from Sigma-Aldrich (MO, USA); HPLC grade methanol (MeOH), chloroform, acetone, acetonitrile (ACN), acetic acid and formic acid (FA) were purchased

from

Fisher

Scientific

(NJ,

USA);

benzoic

acid

(BZAC),

2',4'-difluoroacetophenone (DFAP) and 1-allyl piperazine (ALPZ) were purchased from J&K Chemical Ltd. (Beijing, China); pure water was purchased from Wahaha (Hangzhou, China). Human blood and urine were provided by healthy volunteers in accordance with the requirements of medical ethics; fresh milk was purchased from a local supermarket (Beijing, China); soil samples were collected from the campus of Beijing Institute of Technology (Beijing, China).

Preparation of MIM The MIM was prepared according to the published procedure.43 Briefly, the membrane was activated by being soaked in 3% NaOH (aq.) at 60 °C for 12 h. After water wash, the membrane was soaked in a solution containing 10% AA and 1% KPS at 70 °C for 12 h. Next, the activated membrane was dried after being soaked sequentially in pure water for 1 h; ACN 30 min; and then 0.15 M AIBN ACN solution for 20 min.

Sample preparation For a standard compound, the prepolymer solution for molecularly imprinting was prepared by dissolving 1 mmol the corresponding template molecule and 6 mmol MAA in the mixture of chloroform and MeOH (5:1, v/v). The solution was fully



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mixed and kept at room temperature for 1 h to achieve complete prepolymerization. And then, 30 mmol EGDMA and 2 mmol AIBN were dissolved in the solution. The activated membrane was immerged in the solution, and deoxygenized in an ultrasonic bath for 10 min and then kept in room temperature for 2 h. Next, the membrane was heated under nitrogen protection at 65 °C for 24 h in an oven. When the reaction was completed, template molecules were removed repeatedly by the mixture of MeOH and acetic acid (10:1, v/v). Finally, the MIM was rinsed by pure water to neutral. MIM was typically prepared beforehand, and kept in a desiccator and ready for use. To prepare practical samples, different procedures were carried out based on the sample sources. CLE, MTX CPFX were directly spiked into human urine, blood and milk, respectively. Soil samples were sieved first to remove large pieces of stones and clods. MeOH solution spiked with CPF was put into the soil sample (1.5 mL MeOH used for 100 mg soil), and shook well before using. The MIM was then shaken in the 1.5 mL simulated real sample solution for 20 min at room temperature in a 2 mL EP tube. After being rinsed by pure water and dried out at room temperature, the MIM was ready to use in the MIM-ESI MS experiment. In nanoESI-MS experiments, 0.5 mL milk sample was lyophilized, and then was extracted by 0.5 mL MeOH with 0.1% FA.

Mass spectrometry analyses Experimental conditions for mass spectrometry analyses coupled with different ionization techniques were briefly described as follows, and the detailed conditions


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were given in Supporting Information. (Supporting Information Available: [detailed experimental conditions] This material is available free of charge via the Internet at http://pubs.acs.org). MIM-ESI-MS: All experiments were carried out using a Bruker HCT mass spectrometry (Bruker Daltonics Inc., MA, Germany). Nitrogen was used as drying gas (flow rate, 10 L⋅min-1; temperature, 150 °C). The capillary voltage was set at -1.0 kV for positive ion mode. In a MIM-ESI-MS experiment (Figure 1), MIM (~ 5 mm equilateral triangle) with the enriched analyte was fixed by a copper clip and kept the tip of the membrane ~5 mm away from the MS inlet. A high voltage (HV) of +2.5 kV and 8 µL elution solvent (0.1% FA in MeOH) were applied onto the MIM. The process of HV optimization was described in Section 2 of Supporting Information (Supporting Information Available: [Optimization of applied HV on MIM] This material is available free of charge via the Internet at http://pubs.acs.org) Nanoelectrospray-MS: The capillary voltage was set at -1.2 kV for positive mode. Distance between nanospray tip and MS inlet was set at ~ 5 mm. PS-MS: The sample (2 µL) was loaded directly onto a triangular section of chromatography paper (about 6 mm base width and 6 mm height). Spray voltage was set at 4.5 kV, and 8 µL MeOH with 0.1% FA was used as elution solvent.

3. RESULTS AND DISCUSSION

MIM-ESI was developed following our previous work (MESI) coupled with the concept of molecular imprinting (MI). AA was used as the functional monomer in this



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study for decreasing non-specific binding in MIM synthesis.41 The molecularly imprinted membrane (MIM) was synthesized and then characterized to confirm its surface modification (Figure 2). Using RAC as the example template molecule, scanning electron microscopy (SEM; SU8010, Hitachi, Japan) was used to examine the surface microstructures of blank PVDF membrane, RAC−MIM and the MIM after removing RAC, respectively (Figure 2a-2c). In the SEM image of blank PVDF membrane (Figure 2a), the thin fiber could be observed clearly. The reticular fiber was filled when RAC was cross-linked onto the membrane (Figure 2b). Finally, the fiber structure on the membrane was recovered after removing RAC (Figure 2c). Besides SEM, infrared (IR; Tensor II, Bruker, Germany) spectra of these membrane samples were measured (Figure 2d-2f), and vibrations of C−F in 1149, 1159 and 1139 cm-1 were able to be found in all of the three samples, respectively. RAC-MIM revealed carbonyl group (−C=O) and carboxyl group (−CO2H) infrared absorption in 1698 and 2945 cm-1, indicating that the molecular imprinted layer has been cross-linked on the membrane (Figure 2e). As shown in Figure 2f, intensities of characteristic peaks of RAC at 2951 and 1719 cm-1 decreased significantly, suggesting that most of the template molecules were removed from the MIM. However, there was still a small amount of template molecules remained on the membrane. Therefore, in our experiments, target analytes were not used directly as the templates, but molecules which have similar structures but different molecular weights. A series of experiments were carried out to verify characteristics of synthesized


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MIM (Figure S2). Fresh milk spiked with ciprofloxacin (CPFX) was used as the model sample, and the different concentrations (high concentration, 1 µg⋅mL-1; medium concentration, 10 ng⋅mL-1; low concentration, 1 ng⋅mL-1) were investigated, respectively. Specific recognition function was demonstrated by the comparative experiments using molecularly imprinted membrane (MIM) versus nonimprinted membrane (NIM). Also, relationships between the membrane surface properties and the molecular imprinting function were investigated, including the comparison of hydrophilic and hydrophobic PVDF membrane, different substrate materials of membrane, the activation step in the process of MIM synthesis. In addition, the unique selectivity of MIM was investigated (Figure S3). Detailed information was given in Section 3 of Supporting Information (Supporting Information Available: [Verification of MIM properties] This material is available free of charge via the Internet at http://pubs.acs.org). These results demonstrated that the synthesized MIM layer plays key role in target enrichment when it has been cross-linked onto membrane. This enrichment mechanism is different from the reported cases.44,45 MIM-ESI has similar ionization mechanism to that of MESI and PS. With the capability of target analyte enrichment, MIM-ESI is expected to be able to achieve lower detection/quantitation limits of analytes than other conventional ionization techniques. As an example, Figure 3 showed the linear quantitation ranges for CPFX using different ionization techniques, including MIM-ESI, PS and nanoESI. In MIM-ESI, NFLX was used as the template molecule, and 8 µL of MeOH with 0.1% FA was used as the elution solvent. MESI cannot be tested in this experiment because



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its dialysis membrane is not able to handle elution solvent containing high ratio of organic phase. A limit of quantitation (LOQ) of 1 ng⋅mL-1 was obtained using MIM-ESI, compared to 50 ng⋅mL-1 and 10 ng⋅mL-1 when using nanoESI and PS, respectively. MS and MS2 spectra at the LOQs in each case were also plotted in Figure 3b-3f. It is believed that the molecular imprinting technique could effectively enrich target molecules and especially effective for low concentration analytes in complex matrices. As an ambient ionization method, MIM-ESI could be applied to the direct quantification of target analytes in complex samples. As a first example, CLE was quantified in urine samples (Figure 4). CLE is a β-adrenergic agonist. Pigs treated with CLE may pose potential risk and result in a series of harmful effects on consumer health.46,47 In our study, CLE samples were prepared by spiking CLE powder into human urine (to simulate porcine urine), and human urine samples without spiking CLE were used as blank samples. RAC was used as the template molecule in preparing the MIM, and all samples were analyzed using MIM-ESI-MS without any sample pretreatment. Figure 4a plots the calibration curve of CLE in urine, which was linear over the range of 0.1−100,000 ng⋅mL-1 with a correlation coefficient (r2) of 0.9973. Due to the complexity of the sample, tandem MS was used for CLE quantitation. The LOD (SNR = 5) and LOQ (SNR = 15) were 0.02 and 0.1 ng⋅mL-1, respectively. Figure 4b showed the MS spectrum of CLE (m/z 277) in the urine sample at a concentration of 0.1 ng⋅mL-1, and CLE was confirmed by its product ions of m/z 259 and m/z 203 as shown in the tandem MS spectrum (Figure 4c). As a


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control experiment, m/z 259 and 203 were not able to be observed in blank samples (Figure 4d). Besides food safety applications, the uses of MIM-ESI MS in different application fields were also demonstrated, such as quantifications of drug concentrations in blood, pesticide in soil and antibiotic residues in milk. MTX in blood, CPF in soil and CPFX in milk were analyzed by MIM-ESI MS, and TMP, TCFM and NFLX were applied as template molecules, respectively. Linear range of quantitation and the corresponding tandem mass spectra at LOQs were shown in Figure 5a-5c and 5d-5f, respectively. By enriching target analytes on membranes through the molecular imprinting technique, low quantification limits were achieved, which are 0.5 ng⋅mL-1, 1 ng⋅mL-1 and 1 ng⋅mL-1 for MTX in blood, CPF in soil and CPFX in milk, respectively. Detailed information for methodology validation was summarized in Supporting Information (Supporting Information Available: [methodology validation] This material is available free of charge via the Internet at http://pubs.acs.org).

4. CONCLUSIONS In summary, molecularly imprinted technology was applied onto membrane electrospray ionization as a ambient ionization technique with enhanced sensitivity. MIM-ESI MS has satisfactory performances for directly rapid quantification of targeted analytes in complex samples. This technique shows enhanced sensitivity for various analyses. With the capability of quantifying low concentration analytes in



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complex samples, MIM-ESI MS could potentially be very useful for multiple applications.

ACKNOWLEDGEMENTS This research was supported by NSFC (21475010, 81471919), MOST China (2016YFC1202700) and Beijing NSF (16L00065).

SUPPORTING INFORMATION This material is available free of charge via the Internet at http://pubs.acs.org.


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Figure 1. The workflow and analyte ionization in MIM-ESI.



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Figure 2. Characterization of synthesized molecularly imprinted membrane (MIM): a−c) Scanning electron microscopy (SEM) photos of membrane samples: the blank PVDF membrane, RAC cross-linked onto the MIM and MIM after removing RAC; d−f) IR spectra of membrane samples: the blank PVDF membrane, RAC cross-linked onto the MIM and MIM after removing RAC.


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CPFX

c)

MIM-ESI (NFLX−CPFX) nanoESI PS

5

×104

CPFX, 50 ng⋅⋅mL-1 by nanoESI

e)

4 3

×104

CPFX, 10 ng⋅⋅mL-1 by PS

[M+H]+

1.2 Intensity

Log10 Intensity

6

8

332 Intensity

a)

0.6

[M+H]+

4

332

2 0 1 2 3 4 Log10[conc. of CPFX (ng⋅mL-1)]

6

Intensity

8

3

313

332

m/z 0 100

150

×103

200

1.2

[M+H]+

0 100 150 200 250 300 350

250

300

0 100

350

150

f)

[M-CO2H+H]+

250

×103

287 6

CID

[M-F+H]+

313

0.6

200

300

[M+H]+

[M-CO2H+H]+

287

332 CID

[M-F+H]+

3

313

332

[M+H]+

332

332 250

m/z

300

350

350

m/z

332

[M-CO2H+H]+ [M-F+H]+

4

d)

287

CID

200

m/z

CPFX, 1.0 ng⋅⋅mL-1 by MIM-ESI 332

150

Intensity

×104 ×102

0 100

Intensity

b)

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 100

150

200

250

300

350

m/z

0 100

150

200

250

300

350

m/z

Figure 3. Linear quantitation ranges and tandem MS spectra of CPFX using different ionization techniques: a) standard curves of CPFX in milk samples detected by different ionization methods; b) MS and MS2 spectra in LOQ of CPFX in milk samples detected by MIM-ESI-MS; c−d) MS and MS2 spectra in LOQ of CPFX in milk samples detected by nanoESI-MS; e−f) MS and MS2 spectra in LOQ of CPFX in milk samples detected by PS-MS.



a)

×106

b) ×104

4

0.1 ng⋅⋅mL-1 CLE in urine

4

LOQ: 0.1 ng⋅mL-1

Intensity

Intensity

CLE

2

2

[M+H]+

277

y = 48.906x + 90804 r² = 0.9971

0 0

0 100 150

1.0 ×105

0.5

×104 1.0

277 CID

d)

[M-H2O+H]+ 259

277

3

0.5 [M+H]+

203 0 100 150

200

300

300

350

266 256

2

265 148

350

275

CID

1

277 250

250

Blank sample

×102

Intensity

c)

200

m/z

Conc. of CLE (ng⋅mL-1)

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 25

0 100 150

m/z

207 200

250

277 300

350

m/z

Figure 4. CLE quantification in urine samples by MIM-ESI-MS: a) the calibration curve of CLE in urine; b) MS spectrum of CLE in the urine sample at a concentration of 0.1 ng⋅mL-1; c) MS2 of CLE at a concentration of 0.1 ng⋅mL-1; d) MS2 spectrum of the corresponding blank samples.


Page 19 of 25

Drug Concentration in Blood

Pesticide Residues

Antibiotic Residues

TMP−MTX in blood

TCFM−CPF in soil

NFLX−CPFX in milk

a) ×10

5

6

b) ×10 1.5

MTX

3

c)

CPF

×106

CPFX

y = 27.967x + 10543 r² = 0.9960

0

0 0

y = 13.838x + 21696 r² = 0.9985

1.0 ×105

0.5

0

Conc. of MTX (ng⋅mL-1)

4

e) ×102

455 308 CID

MTX 0.5 ng⋅mL-1 455

300

400

m/z

500

f)

600

×102

[M-CH3+H]+

8

350

[M+H]+

350 CID

4 CPF 1.0 ng⋅mL-1

[M+H]+

0 200

0

0 100

200

1

2

3

4

332 CID

287 [M-CO2H+H]+

CPFX -1 4 1.0 ng⋅mL 313

[M-F+H]+

300

400

5 ×105

Conc. of CPFX (ng⋅mL-1)

335

8

[M-Glu+H]+

2

1.0 ×105

0.5

y = 250.51x + 12193 r² = 0.9992

Conc. of CPF (ng⋅mL-1)

Intensity

d) ×102

0.5

LOQ: 1.0 ng⋅mL-1

0

Intensity

1

1.0 LOQ: 1.0 ng⋅mL-1 0.5

Intensity

LOQ: 2 0.5 ng⋅mL-1

Intensity

Intensity

1.0

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


500

m/z

0 100

150

200

250

[M+H]+

332 300 350

m/z

Figure 5. The uses of MIM-ESI MS in different application fields,including quantifications of drug concentrations in blood, pesticide residues in soil and antibiotic residues in milk: a−c) calibration curves of MTX in blood, CPF in soil and CPFX in milk; d−f) MS2 spectra of LOQs of MTX, CPF and CPFX.



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TOC


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Molecularly Imprinted Membrane Electrospray Ionization for Direct

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