Mass Spectrometry for Paper-based Immunoassays: Towards Ondemand

Transcription

1 Supporting Information for Mass Spectrometry for Paper-based Immunoassays: Towards Ondemand Diagnosis Suming Chen 1,2, Qiongqiong Wan 1,2 & Abraham K. Badu-Tawiah 1,* 1 Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 4321 USA 2 These authors contributed equally to this work Correspondence should be addressed to A.B-T. (badu-tawiah.1@osu.edu) S1

2 Material Whatman No. 1 chromatography paper, lyophilized bovine serum albumin (BSA) and glycerol were purchased from VWR (Radnor, PA, USA). Gel blot paper (GB3, 15 cm 2 cm) was obtained from Whatman, Inc. (Sanford, ME, USA). (Carboxymethyl)trimethylammonium hydrochloride, (3- carboxypropyl)trimethylammonium chloride, Potassium periodate, 1X phosphate buffered saline (PBS), 2,4-dinitrophenylhydrazine, and sterile-filtered US-origin human serum (from human male AB plasma), Tween 2 (for molecular biology), and Greiner high and medium binding 96 well plates were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. 4-(2-Hydroxyethyl)phenyl isothiocyanate was purchased from Organix Inc (Woburn, MA, USA). Tris(hydroxymethyl)aminomethane (Tris) and sodium chloride were purchased via VWR from Avantor Performance Materials (Center Valley, PA, USA), respectively. Lyophilized Plasmodium falciparum histidine-rich protein 2 (PfHRP2, 37 kda) was purchased from CTK Biotech (San Diego, CA, USA). The cancer antigen 125 (CA-125) and carcinoembryonic antigen (CEA) were purchased from LEE Biosolutions (Maryland Heights, MO, USA). The anti-malaria PfHRP2 IgG monoclonal antibodies (ABMAL-444 (Clone 44) and ABMAL-445 (Clone 45)), anti-ca 125 monoclonal antibodies (capture and detection), and anti-human CEA monoclonal antibodies (capture and detection) were purchased from Arista Biologicals Inc. (Allentown, PA, USA). ABMAL- 444 (Clone 44) was used as the capture antibody and ABMAL-445 (Clone 45) was used as the detection antibody. 1 mg lyophilized PfHRP2 was dissolved in 2 μl of 2% PBSA to get 64 μm (5 mg/ml) stock solutions. 1.5 μl aliquots of the above solution were stored at -8 C. The capture antibody, as received from the manufacturer, was stored in 1 μl aliquots at -2 C. UltraCruz TM Micro G-25 Spin Columns were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). HRP-conjugated anti- PfHRP2 mouse monoclonal and 3,3,5,5 -tetramethylbenzidine (TMB) ELISA substrate were purchased from Abcam (Cambridge, MA, USA). Mass spectrometry analysis All experiments were carried out with a Velos Pro ion trap mass spectrometer (Thermo Scientific, San Jose, CA, USA), operated in the full mass spectrum mode or selected reaction monitoring (SRM) mode, except the MS characterization of the probe-conjugated detection antibody that was done on Orbitrap mass spectrometer (Exactive Plus EMR, Thermo Scientific, San Jose, CA, USA). The specific product ions produced by collision-induced dissociation (CID) were monitored. The Xcalibur software (version 2.2) was used for control of the MS system and data acquisition. Argon gas (99.995% purity) was used as collision gas. The temperature of the MS inlet capillary was 25 C. The nano-electrospray ionization (nesi) capillary were pulled from borosilicate glass capillaries with filament (Sutter Instrument, USA) using a micropipette puller (Model P-97, Sutter S2

3 Instrument Co., Novato. CA, USA). The parameters for nesi, TPS and ESI: full MS mass range: 5-1 Da, MS/MS mass range: 5-5 Da; maximum ion injection time: 1 ms, microscan time: 3 μs. The optimal normalized collision energy for CID is 25-3%. For the nesi MS experiment, the distance between the tip of capillary and MS inlet was 8 mm. The flow rate is 3 μl/min for ESI MS experiments. Touch paper spray mass spectrometry analysis For the TPS MS analysis, a second wax-printed substrate (i.e., the paper spray strip) with sharp triangular tips was stacked or placed under the reaction strip. Then, a metal clamp was used to fix the final double-layer paper scaffold and placed in front of the mass spectrometer. The distance between the paper spray tip and MS inlet was 5 mm. Upon the addition of 5 μl of ACN-H2O (1:1, v/v) and application of 4 kv of voltage, the analytes on the top were extracted, penetrate down to the bottom paper and reached the MS inlet by means of charged electrosprayed micro-droplets. The MS signals were recorded by the mass spectrometer. The elution and transfer efficiency of the dried deposited probes from the paper substrate were investigated using a previously reported protocol for paper spray MS, with slight modification. [1] Specifically, solutions of CMTA/CPTA (5 μl, 1 nm) in ACN-H2O (1:1, v/v) were first pre-deposited onto blank paper test (circular) zones and dried in the open air. Then, we attached the second paper spray strip substrate to the bottom layer of first paper for TPS MS/MS detection. 5 μl of ACN-H2O solution was added to the dried reagent, and the application of DV voltage enabled the extracted ion chromatograms (XIC) of CMTA ( 14 96) and CPTA ( ) to be recorded. After the signal decreased to a minimum, the process was repeated five consecutive times from the same test zone bearing a single sample using 5 μl of ACN-H2O for each of the five extraction events. The percentages of each eluted peak of CMTA and CPTA in XIC for these five elution steps were calculated to characterize the elution and transfer efficiency. Percentage of peak area (%) = An / (A1 + A2 + A3 + A4 + A5). Peak areas are calculated by Origin software (version 8.). Calculation of the limit-of-detection The minimum concentration of antigen solution that gives an average MS intensity ratio that is higher than the average intensity ratio from the negative surface by three times the standard deviation of the mean. S3

4 Synthesis of the ionic probes As shown in Figure S1, the first step of the probe synthesis is the preparation of the acyl chloride. Anhydrous (carboxymethyl)trimethylammonium hydrochloride (CMTA, 1, 2 mg, 1.38 mmol) or (3-carboxypropyl)trimethylammonium chloride (CPTA, 5, 2 mg, 1.24 mmol) was suspended in 8 ml (.11 mol) of thionyl chloride in a round bottomed flask. The reaction system was heated at 6 C in a water bath for 1 h. After the emission of sulfur dioxide ceased, thionyl chloride in excess was then removed by evaporation under reduced pressure. The product was washed four times with 1 ml of n-hexane with subsequent removal of the solvent by decantation. The crude product was dried in vacuum to afford 21 mg of acyl chloride 2 or 22 mg of 6 in white powder. The acyl chloride 2 (189 mg, 1.16 mmol,) or 6 (11 mg, 1.16 mmol), and 4-(2- Hydroxyethyl)phenyl isothiocyanate (3, 165 mg,.922 mmol) were mixed in anhydrous acetonitrile solvent (2 ml). The reaction mixture was heated up to 5 C and stirred for 6 h in a water bath, after which the reaction mixture was cooled to room temperature. After removal of the precipitate from the bottom, the solvent of the supernatant was removed under reduced pressure. The crude product was purified by recrystallization in acetonitrile solvent. The final products of 2-(4-isothiocyanatophenethoxy)-N,N,N-trimethyl-2-oxoethanaminium chloride (ITEA, 4, 188 mg, 48% yield) and 4-(4-isothiocyanatophenethoxy)-N,N,N-trimethyl-4-oxobutan- 1- aminium chloride (ITBA, 7, 114 mg, 36% yield) were obtained with light yellow powder. (4) 1 H NMR (4 MHz, CD3CN) δ (m, 4H), 4.42 (t, 4H, J = 6.8 Hz), 3.27 (s, 9H), 2.98 (t, 2H, J = 6.8 Hz). 13 C NMR (1 MHz, CD3CN) δ 164.6, 137.8, 13.5, 125.9, 66., 62.9, 53.6, HRMS-ESI (): [M-Cl] + calcd for C14H19N2O2S +, ; found, (7) 1 H NMR (4 MHz, CD3CN) δ (m, 4H), 4.29 (t, 2H, J = 6.6 Hz), (m, 2H), 3.4 (s, 9H), 2.95 (t, 2H, J = 6.6), 2.35 (t, 2H, J = 6.6 Hz), 1.97 (m, 2H). 13 C NMR (1 MHz, CD3CN) δ 172.4, 139.1, 131.1, 126.4, 65.9, 65.1, 53.4, 34.8, 3.6, HRMS-ESI (): [M-Cl] + calculated for C16H23N2O2S +, ; found, Preparation of aldehyde-functionalized paper Paper with aldehyde functional groups was prepared by soaking sheets (3 8 ) of Whatman No. 1 chromatography paper in a.3 M KIO4 solution at 65 C for 2 hours (Figure S2a). [2] After the reaction, the sheets were washed three times by dipping them in fresh deionized water (dih2o) for one minute each and pouring off the water at the end. After the last wash had been poured out, the sheets were blotted with paper towels and dried in a desiccator for at least 12 hours. A wax mask containing circular wax-free regions (3 mm in diameter) was printed on them using a solid ink printer set to the default parameters for photo-quality printing. The printed sheets were S4

5 placed in an oven (15 C) for 9 seconds. As a result of the heat, the wax melted and spread through the thickness of the paper [3] and created circular (2 mm in diameter) hydrophilic test zones separated by hydrophobic wax barriers (Figure S2b). The presence of the aldehyde groups in the test zones allowed us to covalently immobilize amine-containing molecules (such as the anti-pfhrp2 capture antibody in this study) to the surface of the paper through a Schiff-base linkage (Figure S2c). The sheets of aldehyde-functionalized paper were stored in a desiccator until use. Dinitrophenylhydrazine (2,4-DNP) test The solution of 2,4-DNP, used as a chemical reagent to test the presence of the aldehyde groups in paper was prepared by dissolving 2,4-dinitrophenylhydrazine (3 mg) in concentrated sulfuric acid (1.5 ml). The solution was then added, with stirring, to a mixture of water (2 ml) and ethanol (7 ml). The resulting solution was mixed thoroughly. For the test, 3 μl of the above solution was added to each test zone of untreated and aldehyde-functionalized paper and incubated for 1 min. As a control, 3 μl of water was added to the paper strips without 2,4-DNP. After the incubation, the remaining solution of 2,4-DNP on the surface was wicked by bringing the bottom of the test zone in contact with a blotting paper. Each test zone was then washed with 15 μl of water (three washes of 5 μl each (3 5 μl)) by adding the wash solution to the top of the test zone and pressing the bottom surface against a blotting paper to wick the solution. The rinsed paper was allowed to air dry at room temperature for 5 min and then observed the color of each test zone. Preparation of probe-conjugated detection antibody The method of conjugation of the ionic probes [2-(4-isothiocyanatophenethoxy)-N,N,N- trimethyl- 2-oxoethanaminium chloride (ITEA) and 4-(4-isothiocyanatophenethoxy)-N,N,N- trimethyl-4- oxobutan-1-aminium chloride (ITBA)] to proteins is described below. ITEA or ITBA was dissolved in PBS buffer solution to form the 2 mm probe solution. 1 μl of the above solution was mixed with a 2 μl solution (5.57 mg/ml) of the detection antibody in 1 mm PBS buffer (ph 7.4) to give a total reaction volume of 21 μl for ITEA conjugation. The buffer was exchanged to.1 M NaHCO3 (ph 8.5) for ITBA conjugation. The reaction mixture was placed at 4 C overnight for ITEA conjugation and 4 h for ITBA conjugation. During the reaction, the isothiocyanate functional group of the probe reacts with the amine group of the lysine residues of the antibody to form a thiourea bond (Figure 1d). At the end of the reaction, the excess probe was separated from the probeconjugated detection antibody by size-exclusion columns with a Sephadex matrix (PD-1 Desalting Column and Micro G-25 Spin-Column). Then the buffer was exchanged back to PBS for ITBAconjugated antibody. The purified and characterized probe-conjugated detection antibody was S5

6 diluted to make 5% v/v glycerol stock and stored in 1 μl aliquots at -2 C until use. In order to determine the average number of probe molecules coupled to each detection antibody molecule, quantification by MS was combined with UV visible absorbance spectroscopy. Because of the much overlap of absorption spectra between probe and antibody, the ratio of probe to antibody can t be calculated based only on absorption spectroscopic data (Figure S14). The detailed steps are described as following: 1) Measurement of the concentration of the conjugated probe. Calibration curves were first plotted for (carboxymethyl)trimethylammonium hydrochloride (CMTA) and (3-carboxypropyl) trimethylammonium chloride (CPTA) by nesi MS/MS (Figure S9). Then NH4OH solution (final concentration.1 M for ITEA, and 1 M for ITBA) was added to the diluted solution of purified probe-conjugated antibody to hydrolyze the labeled ITEA (2 min) or ITBA (2 min) for generating the charge-tag products CMTA or CPTA. After the hydrolysis, 1 nm (final concentration) of CPTA and 1 nm (final concentration) of CMTA were added to the hydrolyzed solution of ITEAconjugated antibody and ITBA-conjugated antibody, respectively as internal standards. The solutions were subsequently subjected to nesi MS/MS analysis, and the concentration of CMTA or CPTA in probe-conjugated solution can be calculated based on the intensity ratio of product ions 87 and 59 and equations of linear regression. The concentrations of hydrolytic products CMTA or CPTA represent the correct concentrations of conjugated ITEA or ITBA. 2) Calculation of the absorbance contribution of probes at 28 nm. First, we measured the absorbance of pure ITEA and ITBA solutions, and the molar extinction coefficient of probes were calculated to be ε28 = L mol -1 cm -1. The two probes were proved to have almost the same molar extinction coefficient at 28 nm due to the equal UV absorption moiety. Thus the absorbance contribution of ITEA or ITBA in probe-conjugated antibody at 28 nm can be calculated. 3) Calculation of the molar ratio of probe and antibody. After calculating the absorbance contribution of probe at 28 nm, the correct concentration of conjugated antibody (ε28 = L mol -1 cm -1 ) could be calculated by subtracting the absorption contribution of probes from the total absorbance value at 28 nm (A28). Then the molar ratio (R) of probe and antibody can be obtained based on each real molar concentration (C) as shown in the following equation: R = C probe C antibody = C probe 28 A 28 C probe ε probe 28 ε antibody The labeling ratio is about 1/dAb for ITEA-conjugation and 2/dAb for ITBA-conjugation. Capture of PfHRP2 from biofluid onto paper S6

7 The hydrophilic test zones of the aldehyde-functionalized paper were used for the detection of PfHRP2 using a sandwich immunoassay. [4] For ease-of-use, strips of paper containing four test zones each (2.8 cm 1.5 cm) were cut from the sheets of the previously oxidized chromatography paper. We used a flow-through system [5] where both the top and the bottom surfaces of the test zones were open to the atmosphere. Therefore, during the incubation steps, the paper strips had to be suspended in air to prevent wicking of the solutions from the test zone. This layout was accomplished by placing each end of a paper strip on the lid of a.5 ml centrifuge tube that was fitted inside the frame of an empty pipette-tip box. Each tube extended upwards from the frame and created a raised support for the paper strip. The test zones on each paper strip were thus completely suspended in air between two supports. Multiple centrifuge tubes were fitted into one pipette-tip box to enable simultaneous incubation of 1 strips of paper (4 test zones) in every box. The boxes were kept humid by partially filling them with dih2o and keeping their lids closed during the incubation steps. The test zones to be prepared for the immunoassay were placed on supports, as described above, inside a humid pipette-tip box. A stock solution of the capture antibody was diluted to 67 μm (1 mg/ml) using 1X PBS and glycerol was added to a final concentration of 1%v/v. 2 μl of the above solution was added to each test zone and incubated overnight. After the incubation, the remaining solution of the capture antibody on the surface was wicked by bringing the bottom of the test zone in contact with a blotting paper. Each test zone was then washed with 4 μl of 1X PBS (two washes of 2 μl each (2 2μL)) by adding the wash solution to the top of the test zone and pressing the bottom surface against a blotting paper to wick the solution. To block the excess aldehyde groups against non-specific binding of proteins, each test zone was then incubated with 1 μl of 1X TBS (Tris-buffered saline) in a humid pipettetip box. The excess solution was wicked on the blotting paper and the test zones were washed with 4 μl of 1X PBS (2 2 μl). Following the wash, 2 μl solution of PfHRP2 (prepared in either 1% PBSA (1%w/v BSA in 1X PBS) or human serum) was pipetted on a test zone and incubated in a humid pipette-tip box for 3 minutes. For experiments in buffer, PfHRP2 solutions were prepared by adding 1.5 µl stock solution of PfHRP2 to 22.5 µl of 1% PBSA to make a 1 μm solution, and then diluted further with 1% PBSA to get the lower concentrations. For experiments in human serum, the solutions were prepared as above using undiluted serum instead of PBSA. For negative control, a test zone was incubated with 2 μl of either 1% PBSA or undiluted human serum, without any PfHRP2, for the same duration. At the end of the incubation, the excess solution was wicked on a blotting paper and each test zone was washed with 4 μl of 1X PBS (2 2 μl). S7

8 Detecting the presence of PfHRP2 using ionic probe-conjugated antibody A 33 nm (5 μg/ml) solution of the ITEA- or ITBA-conjugated detection antibody was prepared using 1% PBSA. Each test zone that was contacted with a sample (with or without PfHRP2) was incubated with 5 μl of the above solution in a humid pipette-tip box for 3 minutes. At the end, the excess solution was wicked and each test zone was washed sequentially with 1X PBS (2 2 μl) and dih2o (1 2 μl). After the washing, the remaining solution of the capture antibody on the surface was wicked by bringing the bottom of the test zone in contact with a blotting paper. The dry paper strips could be stored or detected immediately by nesi MS analysis. Multiplexing detection of CA-125 and CEA cancer antigens The hydrophilic test zones of the aldehyde-functionalized paper were also used for the detection of CA-125 and CEA antigens using sandwich immunoassays. The test zones to be prepared for the immunoassay were placed on supports, as described above, inside a humid pipette-tip box. The stock solutions of the capture antibodies CA-125 and CEA were diluted to 1 mg/ml using 1X PBS and glycerol was added to a final concentration of 1%v/v. The solutions were mixed together and 4 μl of the above solution was added to each test zone and incubated overnight. For the control of cross-reactivity testing, 2μL of individual CA-125 and CEA capture antibody solution was added to each test zone and incubated overnight. After the incubation, the remaining solution of the capture antibody on the surface was wicked by bringing the bottom of the test zone in contact with a blotting paper. Each test zone was then washed with 4 μl of 1X PBS (two washes of 2 μl each (2 2μL)) by adding the wash solution to the top of the test zone and pressing the bottom surface against a blotting paper to wick the solution. To block the excess aldehyde groups in the paper test zones, against non-specific binding of proteins, each test zone was then incubated with 1 μl of 1X TBS (Tris-buffered saline) in a humid pipette-tip box. The excess solution was wicked on the blotting paper and the test zones were washed with 4 μl of 1X PBS (2 2 μl). Following the wash, 2 μl mixture solution of CA-125 and CEA antigen with different concentrations (prepared in either 1% PBSA (1%w/v BSA in 1X PBS) or human serum) was pipetted on a test zone and incubated in a humid pipette-tip box for 3 minutes. In the cross-reactivity control, 1 μl of the antigen was added separately to individual test zone with according capture antibody. For experiments in buffer, antigens solutions were prepared by adding 1 µl stock solution of CA-125 and CEA antigens to 1% PBSA to make the 1 ku/ml and 5 μg/ml solutions, and then diluted further with 1% PBSA to get the lower concentrations. For experiments in human serum, the solutions were prepared as above using undiluted serum instead of PBSA. For negative control, a test zone was incubated with 2 μl of either 1% PBSA or undiluted human serum, without any S8

9 antigen, for the same duration. At the end of the incubation, the excess solution was wicked on a blotting paper and each test zone was washed with 4 μl of 1X PBS (2 2 μl). A 5 μg/ml solution of the ITEA- or ITBA-conjugated detection antibody was prepared using 1% PBSA. Each test zone that was contacted with the sample (with or without cancer antigens) was incubated with 1 μl of the mixture solution (test zones for simultaneously detection) or 5 μl of individual solution (test zones for cross-reactivity control testing) in a humid pipette-tip box for 3 minutes. At the end, the excess solution was wicked and each test zone was washed sequentially with 1X PBS (2 2 μl) and dih2o (1 2 μl). After the washing, the remaining solution of the capture antibody on the surface was wicked by bringing the bottom of the test zone in contact with a blotting paper. The dry paper strips could be stored or detected immediately by nesi MS analysis. MS detection of antigen present on the paper surface 1) Nanoelectro spray ionization (nesi) MS detection: An aqueous solution contains 2% of acetonitrile (v/v), NH4OH (.1 M for assay that utilized ITEAlabeled antibody and 1 M for assay that used ITBA-labeled antibody), and internal standard (1 nm CPTA or 1 nm CMTA for immunoassays involving ITEA- or ITBA-labeled antibody, respectively) was prepared. The addition of NH4OH adjusted the ph value of the solution to basic, which catalyzed the hydrolysis of ITEA and ITBA to release the charge-tags CMTA and CPTA quaternary ammonium cations. The acetonitrile solution can accelerate the infiltration of test zones on the paper. In actual experiments, only 2 μl of the above solution was pipetted onto each test zone and incubated in a humid pipette-tip box for a given time (5 min or 2 min for immunoassays involving ITEA- or ITBA-labeled antibody, respectively). After the incubation, the solution on the test zone was transferred to a disposable glass capillary by pipette with capillary pipet tip. Finally, the solution in the capillary was subjected to nesi-ms/ms analysis by applying 1.5 kv voltage through a platinum wire. For the quantitative analysis of PfHRP2 antigen with nesi MS, calibration plots for PfHRP2 spiked in PBS solution and serum by using ITEA- and ITBA conjugated detection antibody were measured. PfHRP2 antigen concentrations are.5,.75, 1, 2.5, 5, 1, 25, and 5 nm for ITEA-conjugated dab. Concentrations are.1,.25,.5,.1,.25,.5, 1, 2.5, 5, 1, and 25 nm for ITBA-conjugated dab. Intensity ratio of product ions from CMTA ( ) and CTPA ( ) were calculated for quantification. 1 nm of CPTA was added as internal standard for ITEA-dAb, and 1 nm of CMTA was added as internal standard for ITBA-dAb. S9

10 2) Touch paper spray (TPS) MS detection: The paper strips completed the immunoreaction were added 5 μl of an aqueous solution contains 2% of acetonitrile (v/v), NH4OH (.1 M for assay that utilized ITEA-labeled antibody and 1 M for assay that used ITBA-labeled antibody), and internal standard (1 nm CPTA or 1 nm CMTA for immunoassays involving ITEA- or ITBA-labeled antibody, respectively) and incubated in a humid pipette-tip box for a given time (5 min or 2 min for immunoassays involving ITEA- or ITBA-labeled antibody, respectively). After the incubation, the paper strips were dried on the air. For the TPS MS analysis, a second wax-printed paper spray strip with sharp triangular tips was stacked or placed under the reaction strip. Then the metal clamp was used to fix the final 3D paper scaffold, consisting of the two 2D layers placed in front of the mass spectrometer. Upon the addition of 5 μl of ACN-H2O (1:1, v/v) and application of 4 kv of voltage, the analytes on the top were extracted, penetrate down to the bottom paper and reached the MS inlet by means of charged electrosprayed microdroplets. The MS signals were recorded by the mass spectrometer. For the quantitative analysis of PfHRP2 antigen with TPS MS, calibration plots for PfHRP2 spiked in PBS solution and serum by using ITEA- and ITBA conjugated detection antibody were measured. PfHRP2 antigen concentrations are.25,.5, 1, 1, 25, and 5 nm for ITEA-conjugated dab. Concentrations are.5,.75,.1, 1, 5, and 1 nm for ITBA-conjugated dab. Intensity ratio of product ions from CMTA ( 14 96) and CTPA ( ) were calculated for quantification. 1 nm of CPTA was added as internal standard for ITEA-dAb, and 1 nm of CMTA was added as internal standard for ITBA-dAb. Each datum point is an average of eight replicates and error bars indicate standard deviation. Storage experiments Storage experiments were performed after the sandwich immunoassay has been completed (i.e., after the capture of antigen, and the binding of the probed-conjugated antibody). These storage experiments were performed to validate the reproducibility of the results after the storage at different times. The concentrations of PfHRP2 antigen are 25 nm and 1 nm for ITEA- and ITBAconjugated antibody involved immunoassays. The experiments themselves were conducted in two ways: Firstly, positive and negative control tests with completed antigen capture and complexation of probe-conjugated antibody were stored for 1, 3, 7, 14, and 3 days. After the specified duration, the test zones were subjected to hydrolysis and release the charge-tags for MS analysis. This stability was also confirmed in the second experiment where the positive and negative control test surfaces were subjected to hydrolysis, immediately after the capture of analyte and probe-conjugated anti-body. The hydrolyzed products were then stored for 1, 3, 7, 14, S1

11 and 3 days. In this case, MS analysis of the dried QUATS was achieved by extracting the cleaved charge-tags for testing. The detailed experimental steps are described below: 1) First, 2 μl of the NH4OH aqueous solution (2% acetonitrile, v/v) containing the internal standard was added in the test zone immediately after the immunoassay. The dried paper stripes, containing the hydrolyzed quaternary ammonium cations were stored in a box at room temperature in the air for up to 3 days. To detect the stored reagents, 2 μl of aqueous solution contains 2 % acetonitrile (v/v) was added onto each test zone for 5 min, after which the solution was transferred into the capillary for nesi MS analysis. For the TPS MS analysis, the immunoassay paper substrate was touched with a paper spray strip and 5 μl of aqueous solution contains 5 % acetonitrile (v/v) was used as elution and spray solution. 2) In a second storage experiment, hydrolysis was not performed immediately after the immunoassay. Instead, the paper stripes were stored directly for specified number of days, after which the stored paper strips were subjected hydrolysis followed by nesi MS or TPS MS detection on-demand. During the time of storage, these paper stripes don t need to be prevented from light. For negative control, the test zone was incubated with undiluted human serum without added PfHRP2 antigen, other conditions are the same. ELISA experiments The ELISA assay of PfHRP2 was performed on the 96-well microplates instead of the paper device, because we want to obtain the best results of this method and then compare with the proposed paper-based MS immunoassay. Firstly, we optimized the capture antibody concentration (1, 2.5, 5, 1 μg/ml) and the detection antibody concentration (.4,.2, 1, 5 μg/ml) with different concentrations of PfHRP2 (5 and 1 ng/ml). Finally, 1 μg/ml of capture antibody and.4 μg/ml of detection antibody were chose because of the best dynamic range and the lowest non-specific background. The detailed steps of ELISA experiment described are below: 1) Dilute the capture antibody to the appropriate concentration (1 μg/ml) allowing sufficient volume for 1 μl per well with coating buffer (.2 M sodium carbonate/biocarbonate, ph 9.4). 2) Add the diluted capture antibody to the plate, cover and incubate overnight at 4 o C. 3) Remove the solution and wash the plate with 2 μl per well wash buffer (25 mm Tris buffer solution, ph 7.2, containing.5% Tween 2) for 3 x 5 minutes on a shaking platform. 4) Add 3 μl blocking buffer (2% BSA in wash buffer) per well, cover the plate and incubate for 1 hour at room temperature. 5) Prepare the samples and standards with diluent buffer (2% BSA in wash buffer). The volume per well should be the same as the capture antibody used in step 1. S11

12 6) Remove the blocking buffer and add the samples and standards. Cover the plate and incubate for 2 hour at room temperature. 7) Remove the solution and wash the plate with 2 μl per well wash buffer for 3 x 5 minutes on a shaking platform. 8) Dilute the horseradish peroxidase (HRP)-conjugated detection antibody to the appropriate concentration (.4 μg/ml). The volume per well should be the same as the capture antibody used in step 1. 9) Add the diluted detection antibody to the plate, cover and incubate for 1 hour at room tempserature. 1) Remove the solution and wash the plate with 2 μl per well wash buffer for 6 x 5 minutes on a shaking platform. 11) Add TMB substrate solution to the plate. The volume per well should be the same as the capture antibody used in step 1. Incubate the plate at room temperature for 15 min, and the desired color intensity can be observed. 12) Stop the reaction by adding an equal amount of stop solution (2 M H2SO4) and measure the absorbance at 45 nm in a microplates reader. Effects of storage, readout time and enzymatic reaction time on ELISA 1) Storage: After the antigen capture (1 ng/ml) and detection antibody complexation, the reaction system was stored by two ways in 96-well plates and kept in dark place: i) in Tris buffer solution (ph 7.2, containing 2% BSA), ii) dry at room temperature. Then the system was stored at room temperature for, 1, 3, 5, 12, 24, 72, 168 h. Finally, 1 μl of TMB substrate solution was added and incubated for 15 min. Upon the addition of 1 μl stop solution, the O.D. values were recorded. 2) Readout time: After the addition of the stop solution, the O.D. values of solution were measured every 5 min until 2 h. 3) Enzymatic reaction time: 1 μl of TMB substrate solution was added into the reaction system that completed the immuoreaction and incubated for 1, 2, 3, 4, 5, 6 and 7 min, respectively. 1 μl of stop solution was added into each solution of different time point and the O.D. values were recorded. S12

13 NCS O O (3) Cl - SOCl 2 Cl N - HO N OH 6 o C, 1 h Cl CH 3 CN, 5 o C, 6 h Cl - N O O NCS Cl - N O OH SOCl 2 6 o C, 1 h Cl - N O Cl HO CH 3 CN, 5 o C, 6 h NCS (3) Cl - N O O NCS Figure S1. Synthesis of the ionic probes. S13

14 Figure S2. Preparation of aldehyde-functionalized hydrophilic test zones on paper for covalent immobilization of amine-containing antibody. a) An aqueous solution of potassium periodate was used to selectively oxidize the C2-C3 vicinal hydroxyl groups in the glucose unit of cellulose to give a dialdehyde product (aldehyde-functionalized paper). b) The modified paper was printed with wax and heated to create hydrophilic test zones surrounded by hydrophobic wax barriers. c) Amine groups on a molecule (e.g. amine groups of the lysine residues of an antibody) can form a Schiff base linkage with the aldehyde groups to covalently immobilize the antibodies on the surface of the paper. S14

15 b) NHNH 2 O 2 N NO 2 + H O NHN R O 2 N NO 2 H R 2,4-dinitrophenylhydrazine aldehyde 2,4-dinitrophenylhydrazone Figure S3. Characterization of aldehyde-functionalized paper using 2,4-dinitrophenylhydrazine (2,4-DNP). a) Colorimetric test results of the untreated and aldehyde-functionalized paper with 2,4-DNP. KIO4-treated aldehyde-functionalized paper reacts to give a yellow color, which indicates the formation of 2,4-dinitrophenylhydrazone, and untreated paper remains a very slight yellow. b) Schematic of the reaction of 2,4-DNP with an aldehyde moiety of the aldehyde-functionalized paper. S15

16 1 Intensity Ratio of 87 / Amount of ITBA-conjugated Antibody (pmol) Figure S4. Evaluation of the binding capacity of the antibody onto the test zone of paper. Typically, 5 μl of.1,.1,.1, 1, 2, 4, and 5 μm (in 1X PBS and glycerol (1%, v/v) solution) of ITBAconjugated detection antibody was added onto the test zones and incubated overnight. The excess solution was wicked on the blotting paper and the test zones were washed with 4 μl of 1X PBS (2 2 μl) and water (2 μl). Then 5 μl of the NH4OH aqueous solution (1 M in 2% acetonitrile, v/v) containing internal standard (CMTA, 1 nm) was added onto each zone for 2 min. Finally, the solution was transferred into the glass capillary and subjected to nesi MS/MS analysis. Intensity ratio of product ions from CTPA ( ) and CMTA ( ) were calculated for quantification. Each datum point is an average of three replicates and error bars indicate standard deviation. The intensity ratio barely increases when the amount of ITBAconjugated antibody is greater than 1 pmol, which signifies that the binding capacity of each test zone. S16

17 CMTA NL: 1.4E6 CPTA NL: 1.5E a) b) 5 1 c) d) CMTA + NH 4 OH NL: 2.8E6 CPTA + NH 4 OH NL: 3.E CMTA NL: 1.E6 1 e) f) 82 CMTA + NaOH NL: 8.2E CPTA NL: 1.9E6 1 g) h) 82 CPTA + NaOH NL: 2.5E Figure S5. The influence of NH4OH and NaOH on the mass spectrometry signals of CMTA and CPTA. a) Mass spectra of pure CMTA in ACN-H2O and b) NH4OH solution; c) Mass spectra of pure CPTA in ACN-H2O and d) NH4OH solution; The ratio of ACN-H2O is 1:1 (v/v), and the concentration NH4OH is 1 M in ACN-H2O (1:1, v/v). e) Mass spectra of pure CMTA in ACN-H2O and f) NaOH S17

18 solution; g) Mass spectra of CPTA in ACN-H2O and h) NaOH solution. The ratio of ACN-H2O is.25:1 (v/v), and the concentration NaOH is.5 M in ACN-H2O (.25:1, v/v). 118 and 14 represent [CMTA] + and [CMTA+Na H] +, respectively, and 146 and 168 represent [CPTA] + and [CPTA+Na H] +, respectively. 6 and 1 may be ascribed to the background ions in the solvent. As can be seen from panel f and h, the presence of NaOH in the solution greatly suppresses the signals of CMTA and CPTA. The MS peaks of CMTA at 118 and CPTA at 146 cannot be observed. The sodium adducts of CMTA at 14 and CPTA at of 168 were also greatly suppressed (insets of panel f and h). In contrast, the presence of NH4OH doesn t have any suppression to the MS signals of CMTA and CPTA. The suppression of NaOH on the MS signals may be similar to the effect reported for nonvolatile salt that changes the efficiency of droplet formation or droplet evaporation, which in turn affects the amount of charged ion in the gas phase that ultimately reaches the detector. [6] In contrast, the volatile NH4OH will not cause this problem a) b) N c) d) N O O 3 O O NCS NCS N N O O 3 OH OH Figure S6. Typical ESI-MS characterization of the ionic probes and their hydrolytic products. (a) ITEA at 279 and (b) its hydrolytic product CMTA at 118. (c) ITBA at 37 and (d) its hydrolytic product CPTA at 146. The concentration of ITEA and ITBA is 1 μm. The hydrolysis solution is.1 M NH4OH for ITEA and 1 M NH4OH for ITBA. 82,1, and 14 are unrelated background ions in the solvent. S18

19 a) b) N N O O O 128 O 162 NCS NCS Figure S7. MS/MS spectra of ITEA and ITBA under collision-induced dissociation conditions. a) MS/MS spectrum of ITEA and the possible fragmentation pathway. b) MS/MS spectrum of ITBA and the possible fragmentation pathway. The concentration of ITEA and ITBA is 1 μm. The normalized collision energy is 3%. S19

20 H CO H 2 O a) b) 87 (CH 3 ) 3 NH + (CH 3 ) 3 N (CH 3 ) 3 N COOH H 2 O Figure S8. MS/MS spectra of CMTA and CPTA under collision induced dissociation. a) MS/MS spectrum of ITEA and the possible fragmentation pathway. b) MS/MS spectrum of ITBA and the possible fragmentation pathway. The concentration of ITEA and ITBA is 1 μm. The normalized collision energy is 28% S2

21 Intensity Ratio of 59 / 87 (1E-3) a) y = 1.38 x R 2 =.99, n = Concentration of CTA (nm) Intensity Ratio of 87 / b) y = 7.24 x +.44 R 2 =.99, n = Concentration of CPTA (nm) Figure S9. Calibration plot for a) CMTA in.1 M NH4OH solution and b) CPTA in 1 M NH4OH solution with ESI MS/MS. 1 nm of CPTA was added as internal standard when detect CMTA, and CMTA was added as internal standard for CPTA detection. Concentrations of CMTA are.1,.5, 1, 5, 1, 5, and 1 nm. Concentrations of CPTA are.1,.5,.1,.5, 1, 5, and 1 nm. Intensity ratio of product ions from CMTA ( ) and CTPA ( ) were calculated for quantification. Each datum point is an average of three replicates and error bars indicate standard deviation. S21

22 Hydrolysis (%) 1 5 a) ph 1 ph 3, 5, 7 ph 11, 12 ph Time (min) Hydrolysis (%) Time (min) Figure S1. Hydrolysis curves of ITEA and ITBA. a) ITEA. b) ITBA. The ph values were adjusted by HCl and NH4OH, concentration of probes is 1 μm. The hydrolysis product was detected and calculated by the ratio of the ion intensity of probes to products in the mass spectra by the equation: hydrolysis (%) = Iproduct / (Iprobe + Iproduct). 1 5 b) ph 12 ph 11 ph 1 ph 3, 5, 7, 9 Supplementary discussion for the hydrolysis of ITEA and ITBA: The core of this performance is the cleavable bond. Compared with other cleavable modes (e.g. photocleavage, oxidation or reduction), the ester linkage can be cleaved simply by changing solution ph (Figure S1). The intact molecular ions of two synthesized probes ITEA and ITBA are easily detected by MS at 279 and 37, respectively (Figure S6). The structures of the charge-tags CMTA ( 118) and CPTA ( 146) released in the presence of NH4OH were confirmed by their unique MS/MS fragmentation patterns (Figure S7,8). As can be elucidated from Figure S1, the proximity of the electron-withdrawing tetramethylammonium cations (CMTA versus CPTA) to the ester bond was observed to influence the hydrolysis rate of the probes. Nearly no hydrolysis was found for both ITEA and ITBA over 1 h at 3 7 ph range. The percent of hydrolysis reached 9% for ITEA (n=1, Figure 1d) at ph 1 for 1 min, but comparable hydrolysis yield for ITBA (n=3) occurred at ph 12. Given the similar structure of these two probes, the difference in hydrolysis rate is thought to be influenced by the distance of induction by the electron-withdrawing ammonium groups. Faster hydrolysis will favor rapid analytical procedure, while the design with longer carbon chain (n) will improve the stability of the probe during test storage. S22

23 1 ITEA ITBA Percentage of Intact Probe (%) Time (Day) Figure S11. Stability of ITEA and ITBA in PBS solution (2 mm, ph 7.4). Each datum point is an average of three replicates and error bars indicate standard deviation. The concentration of the probes is 1 μm. The hydrolysis product was detected and calculated based on the ratio of the ion intensity of probes to products in the mass spectra by the following equation: percentage of intact probe (%) = Iprobe / (Iprobe + Iproduct). S23

24 1 1 a) b) Figure S12. nesi mass spectra of the purified probe-conjugated detection antibody. a) Mass spectrum of ITEA-conjugated antibody upon hydrolysis in.1 M NH4OH solution. The peak at 118 indicates the existence of hydrolytic product CMTA. Inset shows the MS/MS fragmentation of 118. b) Mass spectrum of ITBA-conjugated detection antibody upon hydrolysis in 1 M NH4OH solution. The peak at 146 indicates the existence of hydrolytic product CPTA. Inset shows the MS/MS fragmentation of 146. S24

25 1 a) dab b) ITEA-conjugated dab c) ITBA-conjugated dab S25

26 1 d) dab e) ITEA-conjugated dab f) ITBA-conjugated dab Figure S13. nesi mass spectra of pristine and probe-conjugated antibody detected by high resolution Orbitrap mass spectrometer under denaturing condition using.1% formic acid. a) Full mass spectra of pristine dab, b) ITEA-conjugated dab and c) ITBA-conjugated dab. d)-f) show the enlarged mass range from 288 to 292 with the charge state of 52+. The peaks labeled with red mass numbers come from the conjugated antibodies, which were identified to originate S26

27 from the peaks of pristine antibody. The average mass shifts are 284 Da for ITEA-conjugated antibody and 35 Da for ITBA-conjugated antibody. The molecular weight error with the correct mass increase (+ 1.8 % and.6%) may be ascribed from the slight peaks shift caused by the overlapping of conjugated antibodies with heterogeneously glycosylated antibodies. The conjugation was accomplished at PBS solution (ph 7.4) for 4 h, and concentration of the purified conjugated antibody is 1 μm for nesi MS analysis..2 a) ITEA b).2 ITBA Absorbance.1 Absorbance Wavelength (nm) Wavelength (nm) 1. c) Antibody.6 d) ITEA-conjugated antibody Absorbance.5 Absorbance Wavelength (nm) Wavelength (nm) Figure S14. Absorption spectrum of a) ITEA (1 μm), b) ITBA (1 μm), c) anti-pfhrp2 detection antibody, and d) purified ITEA-conjugated anti-pfhrp2 detection antibody.. S27

28 Intensity Ratio of 59 / 87 (1E-3) 12 a) 8 4 y = x R 2 = Concentration of Pf HRP2 (nm) Intensity Ratio of 59 / 87 (1E-3) b) y = 1.81 x R 2 = Concentration of Pf HRP2 (nm) Intensity Ratio of 87 / 59 2 c) y = x R 2 = Concentration of Pf HRP2 (nm) Intensity Ratio of 87 / d) y = x R 2 = Concentration of Pf HRP2 (nm) Figure S15. Quantitative analysis of PfHRP2 antigen with nesi MS. a)-b) Calibration plot for PfHRP2 spiked in (a) PBS solution and (b) serum by using ITEA-conjugated detection antibody. 1 nm of CPTA was added as internal standard. Concentrations are.5,.75, 1, 2.5, 5, 1, 25, and 5 nm. c)-d) Calibration plot for PfHRP2 spiked in (c) PBS solution and (d) human serum by using ITBAconjugated detection antibody. 1 nm of CMTA was added as internal standard. Concentrations are.1,.25,.5,.1,.25,.5, 1, 2.5, 5, 1, and 25 nm. Intensity ratio of product ions from CMTA ( ) and CTPA ( ) were calculated for quantification. Each datum point is an average of eight replicates and error bars indicate standard deviation. The difference in sensitivity between the two probes may be explained from two aspects: i) the labeling efficiency of ITEA ( 1 per antibody) is lower than ITBA ( 2 per antibody) and ii) the longer linear molecular length of CPTA makes it easier to be fragmented in collision- induced dissociation (CID) and hence producing fragment ions with higher efficiency and intensity. S28

29 Intensity Ratio of 59 / 87 (1E-3) a) Concentration of Pf HRP2 (nm) Intensity Ratio of 59 / 87 (1E-3) b) Concentration of Pf HRP2 (nm) Intensity Ratio of 87 / c) Concentration of Pf HRP2 (nm) Figure S16. Quantitative analysis of PfHRP2 antigen in PBS solution and serum. Intensity ratio of 59/87 versus the concentrations of PfHRP2 spiked in a) PBS solution and b) serum by using ITEA-conjugated detection antibody. 1 nm of (3-carboxypropyl)trimethylammonium chloride (CPTA) was added into the hydrolytic solution as internal standard. Concentrations of PfHRP2 are.5,.1,.5,.75, 1, 2.5, 5, 1, 25, 5, 1, 25 and 5 nm. Intensity ratio of 87/59 versus the concentrations of PfHRP2 spiked in c) PBS solution and d) human serum by using ITBAconjugated detection antibody. 1 nm of (carboxymethyl)trimethylammonium hydrochloride (CMTA) was added into the hydrolyticl solution as internal standard. Concentrations of PfHRP2 are.1,.25,.5,.1,.25,.5, 1, 2.5, 5, 1, 25, 5, 1, 25, 5 nm. Intensity ratio of product ions from CMTA ( ) and CTPA ( ) were calculated for quantification. Each datum point is an average of eight replicates and error bars indicate standard deviation. Intensity Ratio of 87 / d) Concentration of Pf HRP2 (nm) S29

30 O.D. (45 nm) Concentration of Pf HRP2 (ng/ml) Figure S17. Quantitative analysis of PfHRP2 antigen in serum with ELISA method. O.D. represents the absorbance (45 nm) of the solution produced by the enzymatic reaction of horseradish peroxidase (HRP) with 3,3,5,5 -tetramethyl benzidine (TMB) substrate in the sandwich ELISA assay in 96-well microplates. Concentrations are 1, 5, 1, 5, 1, 5 ng/ml. Each datum point is an average of eight replicates and error bars indicate standard deviation. The R 2 value of the curve fit to the datum using the Hill Equation (y = Vmax x n / (k n + x n )) is.98, V = 6.22 ± 3.29, k = ± , n =.74 ±.6. S3

31 1 1 [ITEA] a) b) [ITBA] [CMTA] [CMTA+Na H] + 14 c) d) [CMTA+K H] [CPTA+Na H] Figure S18. Typical touch spray (TSP)-MS characterization of the compounds. (a) Pure ITEA at 279. (b) Pure ITBA at 37. (c) Pure CMTA at 118 ([M] + ), 14 ([M+Na H] + ) and 156 ([M+K H] + ). (d) Pure CPTA at 146 ([M] + ) and 168 ([M+Na H] + ). Concentrations are 1 μm. 15 is unrelated background ions. 5 [CPTA] S31

32 H a) b) (CH 3 ) 3 N 1 H 2 O CO Figure S19. TSP MS/MS spectra of CMTA and CPTA under collision induced dissociation. (a) CMTA of 118 ([M] + ). (b) CMTA of 14 ([M+Na H] + ). c) CPTA of 146 ([M] + ). (d) CPTA of 168 ([M+Na H] + ). Concentrations are 1 μm. The normalized collision energy is 28%. 1 c) d) (CH 3 ) 3 NH + (CH 3 ) 3 N COOH CO 2 S32

33 8 a) Intensity ratio of 96/87 Intensity ratio of 87/ b) y =.642 x -.27 R 2 = Concentration of CTA (nm) y = x R 2 = Concentration of CPTA (nm) Figure S2. Calibration plot for a) CMTA in and b) CPTA with TPS MS/MS. 1 nm of CPTA was added as internal standard when detect CMTA, and 1 nm of CMTA was added as internal standard for CPTA detection. Concentrations of CMTA are.1,.25,.5, 1, 1, 5, and 1 nm. Concentrations of CPTA are.25,.5,.1, 1, 5, and 1 nm. Intensity ratio of product ions from CMTA ( 14 96) and CTPA ( ) were calculated for quantification. Each datum point is an average of three replicates and error bars indicate standard deviation. S33

34 Pencentage of Peak Area (%) Normalized Ion Intensity a) CMTA nd 1st 3rd 4th 5th Time (min) 1 c) CMTA Elution Number Normalized Ion Intensity Pencentage of Peak Area (%) Time (min) st b) d) 2nd 3rd 4th Elution Number CPTA 5th CPTA Figure S21. The elution of CMTA and CPTA from paper substrate recorded by TPS MS. a)-b) The extracted ion chromatograms (XIC) of CMTA ( 14 96) (a) and CPTA ( ) (b) during five elution processes. CMTA/CPTA (5 μl, 1 nm) in ACN-H2O (1:1, v/v) were pre-deposited onto the blank test zones of immunoassay paper substrate and dried at room temperature. The paper spray strip was then attached to the bottom layer of paper for TPS MS detection. 5 μl of ACN-H2O solution was added for elution in each time. The start of elution processes are labeled with red arrows in panels (a) and (b). c)-d) The percentages of each elution peak of CMTA (a) and CPTA (b) in XIC for each of the five elution/extraction steps. Percentage of peak area (%) = An / (A1 + A2 + A3 + A4 + A5). Peak areas were calculated by Origin software (version 8.). S34

35 Normalized Ion Intensity Ratio CMTA 96 / 87 CMTA/CPTA in solution CMTA/CPTA pre-deposited onto paper CPTA 87 / 96 Figure S22. Comparison of the normalized ion intensities of CMTA/CPTA in solution versus predeposited dried samples in the TPS MS analysis. The CPTA (for CMTA analysis) and CMTA (for CPTA analysis) were used as internal standard. For the analysis of CMTA/CPTA in solution, 5 μl of ACN- H2O (1:1, v/v) solution containing CMTA and CPTA (both are 1 nm) was added on the top of TPS paper substrates and subjected to MS/MS analysis. For the analysis of dried CMTA/CPTA samples, 5 μl of CMTA/CPTA (1 nm) in ACN-H2O was pre-deposited onto the blank test zones of the top layer paper substrate and dried at room temperature. Then 5 μl of 1 nm of CPTA (for CMTA analysis) or CMTA (for CPTA analysis) solution in ACN-H2O was added for TPS MS detection. The intensity ratios of the two ions in solution were normalized into total of 1. Intensity ratio of product ions from CMTA ( 14 96) and CPTA ( ) were calculated for quantification. Each datum point is an average of five replicates and error bars indicate standard deviations. S35