Liposome-Enhanced Polymerization-Based Signal Amplification for

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Liposome-Enhanced Polymerization-Based Signal Amplification for Highly Sensitive Naked-Eye Biodetection in Paper-Based Sensors Seunghyeon Kim, and Hadley D. Sikes ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b08125 • Publication Date (Web): 10 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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ACS Applied Materials & Interfaces

Liposome-Enhanced Polymerization-Based Signal Amplification for Highly Sensitive Naked-Eye Biodetection in Paper-Based Sensors Seunghyeon Kim a and Hadley D. Sikes a, b, c* aDepartment

of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

bProgram

in Polymers and Soft Matter, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

cAntimicrobial

Resistance Integrated Research Group, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, Singapore 138602 *E-mail: [email protected]

Keywords: dye-encapsulating liposome; photo-initiated polymerization; colorimetric detection; signal amplification; paper-based diagnostic test

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ABSTRACT

Polymerization-based signal amplification (PBA) is a materials-based approach to improving the sensitivity of paper-based diagnostic tests. Eosin Y is used as an assay label to photo-initiate free radical polymerization to produce colored hydrogels in the presence of target analytes captured by bioactive paper. PBA achieves high-contrast and time-independent signals, but its nanomolar detection limit makes it impractical for early diagnosis of many diseases. In this work, we demonstrated efficient localization of large quantities of eosin Y per captured target analyte by incorporating eosin Y-loaded liposomes into PBA. This new materials approach allowed 30-fold signal enhancement compared to conventional PBA. To further improve the detection limit of liposome-enhanced PBA, we used a continuous flow-through assay format with 100 μL of analyte solution, achieving sub-nanomolar detection limits with high-contrast signals that were easily discernable to the unaided eye.

INTRODUCTION

Paper-based, colorimetric assays have emerged as promising technologies to diagnose diseases in resource-limited settings because they are affordable, quick and easy to perform, and free of laboratory equipment.1,2 Some commercial assays such as rapid diagnostic tests have effectively assisted in early diagnosis and management of falciparum malaria3 and HIV4, but more generally the low sensitivity of colorimetric assays that use gold or dyed polymer nanoparticles as assay labels has limited their use in diagnosis of other diseases.5–9 Therefore, significant interest continues in developing signal amplification techniques for paper-based colorimetric assays to


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improve their sensitivity. Current colorimetric signal amplification methods including silver or gold deposition, horseradish peroxidase conjugation, and catalytic metal coating have achieved 10 to 1000-fold signal enhancement.10–14 However, all of these methods can be subject to timedependent changes in their visual readouts, so they require accurate time-keeping to prevent subjective interpretation of the results by end users.15,16 To address this issue, polymerization-based signal amplification (PBA) has been explored as alternative chemistry for use in paper-based colorimetric assays.17 PBA employs in situ photoinitiated, free radical polymerization on biofunctionalized paper to produce colored hydrogels in the presence of target analytes.17 Eosin Y is used as a photoinitiator following conjugation to reporter proteins that recognize analytes. Eosin Y with triethanolamine and N-vinylpyrrolidone can rapidly remove oxygen via photoredox catalysis,18,19 allowing free radical polymerization to occur only if oxygen is depleted near the paper surface where target analytes have been captured.20 Owing to this threshold process where enough photoredox catalysis has occurred to deplete oxygen and generate colored hydrogels, PBA can provide high-contrast and time-independent signals15,17 with appropriate light intensity and irradiation time.20 Furthermore, PBA is becoming more costeffective and user-friendly as low-cost portable light sources21 and methods for reducing variability in colorimetric results22 are being developed. One major problem of conventional PBA is that the nanomolar limit of detection of analytes it provides15,17,23 is not sensitive enough for early diagnosis of many diseases.24 To improve the detection limit of PBA, many efforts have focused on developing eosin Y-based macroinitiators25,26 to increase the number of the initiators localized per binding event as demonstrated in other biodetection systems using atom transfer radical polymerization,27 redoxinitiated radical polymerization,28 and photo-initiated radical polymerization.29,30 However, poor


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solubility25 and fluorescence self-quenching26 of the eosin Y-based macroinitiators increased nonspecific binding signals and decreased the quantum yield of eosin Y’s triplet state, crucial to photoredox catalysis, limiting the benefits of this approach. This past work motivates the need to deliver highly concentrated and soluble eosin Y to the binding region where analytes have been captured, and to rapidly release the initiators because concentrated eosin Y forms dimers, which are subject to decrease in triplet quantum yield by rapid internal conversion of excited dimers.31 In fact, this idea is analogous to a targeted drug delivery, in which nanocarriers deliver drugs to a specific region within the human body.32 Liposomes, lipid-based vesicles, are often used as the carriers in the drug delivery field due to their biocompatibility, high drug loading efficiency, and rapidly triggered release.33,34 Many well-established technologies for loading both hydrophilic and hydrophobic cargo with high encapsulation efficiency,35–37 narrowing the size distribution of liposomes,38 and reducing non-specific interactions with blood components39,40 have been reported. Furthermore, dye-encapsulating liposomes have been employed as signal enhancement reagents in lateral flow assays.41–44 In light of these features, exploring a targeted eosin Y-delivery with liposomes may yield methods to effectively improve the detection limit of PBA. Here, we synthesized eosin Y-loaded liposomes and incorporated them into PBA to test if a targeted eosin Y-delivery can improve detection limit of PBA. We compared small and large eosin Y-loaded liposomes prepared with different eosin Y-loading conditions to elucidate the relationship between these variables and the sensitivity of PBA. Then, we incorporated liposomes into PBA and confirmed this new materials approach allowed 30-fold signal enhancement compared to conventional PBA using fluorescence microscopy and colorimetric image analysis. To further improve the detection limit of liposome-enhanced PBA, we used a continuous flow-


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through assay format with 100 µL of analyte solution, enabling sub-nanomolar naked-eye biodetection with high-contrast signals.

RESULTS AND DISCUSSION

Scheme 1. Detection of a model protein (streptavidin) using cellulose functionalized with engineered affinity reagents and (A) liposome-enhanced polymerization-based signal amplification (PBA) or (B) conventional PBA. CBD: cellulose binding domain; rcSso7d.SACBD45: CBD-fused, streptavidin (SA) binding variant of reduced charge protein Sso7d from Sulfolobus solfataricus; eosin Y-conjugated streptavidin: eosin 5-isothiocyanate conjugated to streptavidin via isothiocyanate-amine reaction (See Supporting Information).

Liposome-enhanced PBA and conventional PBA


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We developed two different paper-based protein detection assays to compare sensitivity and detection limit of liposome-enhanced PBA and conventional PBA. In these assays, we used a model binding system chosen for its similar thermodynamic binding constant to those of monoclonal antibodies and generalizability to other clinically relevant paper-based immunoassays (further information in SI). As illustrated in Scheme 1, the same amount of the model protein, streptavidin or eosin Y-conjugated streptavidin, is applied to paper functionalized with an engineered molecular recognition reagent, rcSso7d-SA, ensuring that the number of captured streptavidin is the same in both assays. For conventional PBA, the conjugated eosin Y initiates photopolymerization under green light.20 For liposome-enhanced PBA, highly concentrated eosin Y is delivered to captured streptavidin using biotinylated eosin Y-loaded liposomes. The specifically bound liposomes release free eosin Y molecules once monomer solution is added, and the free eosin Y initiates photopolymerization when irradiated with green light. After wicking away unpolymerized monomer solution, phenolphthalein-entrapped hydrogel is generated in both cases so that we can visualize the hydrogel using basic solution. We hypothesized that liposomeenhanced PBA would generate larger amounts of the colored hydrogel than conventional PBA and thus provide higher sensitivity and lower detection limits because the new strategy could increase the rate of radical generation by localizing a greater number of photoactive eosin Y molecules per binding event.


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Figure 1. Preparation and characterization of eosin Y-loaded liposomes. (A) Scheme of preparation of eosin Y-loaded liposomes a, b, and c. See Experimental Section for details. (a) Multilamellar liposomes were extruded through both 400 nm and 50 nm pore membranes sequentially and incubated with 1×eosin Y solution* during dye loading step. (b) Multilamellar liposomes were extruded through 400 nm pore membrane and incubated with 1×eosin Y solution* during dye loading step. (c) Multilamellar liposomes were extruded through 400 nm pore membrane and incubated with 10×eosin Y solution** during dye loading step. (B) Size distribution of eosin Y-loaded liposomes a, b, and c. (C) Mean hydrodynamic radii, % polydispersity, and eosin Y-loading efficiency of the three liposomes. The errors of mean hydrodynamic radii and % polydispersity represent the standard deviations of ten technical replicates. The errors of eosin Yloading efficiency represent the standard deviation of six technical replicates.


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*mol eosin Y : mol phospholipid + mol cholesterol = 1 : 8. **mol eosin Y : mol phospholipid + mol cholesterol = 10 : 8.

Optimization of liposome-based signal enhancement reagents Prior to testing our hypothesis, we optimized assay conditions and engineered eosin Y-loaded liposomes to achieve full potential of liposome-enhanced PBA. Initial investigations indicated that our liposome-based approach yielded high detection limit (~30 nM) due to high non-specific binding signals compared to other paper-based immunoassays (Figure S2). To reduce non-specific interaction between liposomes and rcSso7d.SA-functionalized cellulose surface, we first explored lipid concentration (Figure S3), which is the amount of phospholipids and cholesterol in liposome solution and proportional to liposome concentration. High lipid concentration increased nonspecific binding signals, which could make signal-to-noise ratios small. Low lipid concentration decreased non-specific binding signals, increasing signal-to-noise ratios. However, it also decreased specific binding signals and lowered sensitivity of the assay. Therefore, we chose 0.18 mM of lipid concentration as optimum because it gave the highest signal-to-noise ratio at 300 nM of streptavidin. Then, we added 1% BSA to the liposome solution to reduce collision probability between liposomes and cellulose paper (Figure S4). Using this approach, we could reduce nonspecific binding greatly without decreasing lipid concentration. As a result, we achieved very high signal-to-noise ratios while maintaining high specific binding signals (Figure S5). With the optimized assay, we designed and tested different eosin Y-loaded liposomes to choose the best one that provided the highest specific binding signal and signal-to-noise ratio. Considering that specific binding signals would be positively correlated to the number of eosin Y molecules localized to captured streptavidin, our investigation focused on increasing this number by varying


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liposome size and eosin Y-loading efficiency (Figure 1). As presented in Figure 1A, we started from liposome (a), made larger liposome (b) by using only a 400 nm pore membrane in extrusion step, and loaded more eosin Y into the larger liposome, producing liposome (c). All three liposomes were uniform in size. The small liposome (a) was approximately 100 nm in diameter, and the large liposomes (b and c) were about 280 nm in diameter (Figure 1B-C). Eosin Y-loading efficiency increased when liposome size and eosin Y concentration in the loading step increased (Figure 1C).

Figure 2. Performance of eosin Y-loaded liposomes in streptavidin detection assays. (A) Streptavidin titration curves with background-subtracted mean fluorescence intensity (MFI). Paper-based liposome and conventional assays were performed with a dilution series (0 nM to 300 nM) of streptavidin and eosin Y-conjugated streptavidin (SA-E) respectively as described in Scheme 1. MFI of relevant negative control was used as a background signal for each case. Error bars represent the standard deviation of four independent replicates. (B) Streptavidin titration


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curves with signal-to-noise ratio of MFI. The signal-to-noise ratio of MFI was obtained by calculating the ratio of MFI at each analyte concentration to the relevant background signal. For effective demonstration of the data, we presented results at 0.3 nM to 10 nM of streptavidin concentration. Full version of this plot is available in Figure S6.

To select the best liposome for liposome-enhanced PBA among the three liposomes, we used two indicators: background-subtracted mean fluorescence intensity (MFI) and signal-to-noise ratio of MFI. Background-subtracted MFI can represent specific binding signals because non-specific binding signals from negative control are subtracted from total fluorescence signals. We sought liposomes with higher background-subtracted MFI because it would be more likely to detect lower concentrations of target protein using PBA. Signal-to-noise ratio of MFI turned out to be an important indicator in estimating detection limit of PBA on paper, and it should be at least two to obtain colorimetric signals.17,23 Thus, we sought liposomes showing the signal-to-noise ratio of two at lower concentrations of target protein. As can be seen in Figure 2A, at 3 nM of streptavidin, large liposomes (b and c) generated about the same specific signals as small liposome (a) produced at 30 nM of streptavidin. In terms of signal-to-noise ratio of MFI, small liposome (a) exhibited poor performance compared to large liposomes (b and c) at 0 nM to 10 nM analyte concentration although its small size could reduce non-specific binding signals in paper-based assays (Figure 2B). It is likely because large liposomes delivered much more dye with improved solubility than small one, amplifying specific binding signals significantly. Higher eosin Y-loading efficiency of liposome (c), on the other hand, did not help enhance specific binding signals and rather decreased signal-to-noise ratio of MFI compared to liposome (b) (Figure S6). We attribute these results to a combination of factors such


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as increased non-specific binding signals, fluorescence quenching of highly concentrated eosin Y, and inhibition of streptavidin-biotin interaction by biotin-eosin Y interaction on liposome surface. In light of the aforementioned results, we decided to use the liposome (b) for liposome-enhanced PBA.

Figure 3. Streptavidin titration curves of the optimized liposome assay and the conventional assay in linear range (1 nM to 10 nM of streptavidin). The y-axis represents background-subtracted mean fluorescence intensity (MFI). Error bars represent the standard deviation of four independent replicates. Sensitivity, the slope of each calibration curve,46 was obtained using linear least squares fitting.

Comparison of fluorescence imaging results before PBA To determine how many more eosin Y molecules the liposome-based approach can localize to captured streptavidin than the conventional method, we compared fluorescence imaging results of two streptavidin detection assays before performing PBA. As shown in Figure 2A, specific


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binding signals of the optimized liposome (b) were much higher than eosin Y-conjugated streptavidin (SA-E) at all streptavidin concentrations. It should be noted that specific binding signals of the liposome assay at 3 nM of streptavidin were about the same as the signals generated at 100 nM of streptavidin in the conventional assay. We also compared slopes of the titration curves in linear range to compare the sensitivity of the two methods (Figure 3). The slope of the liposome assay was almost 30 times as large as that of the conventional assay, which is consistent with the previous analysis. Thus, we concluded that the liposome assay could localize approximately 30 times as many photoactive eosin Y molecules as the conventional assay. The extent of signal enhancement in PBA was much smaller than we expected, especially considering that one liposome (b) encapsulated approximately 11,000 times more eosin Y molecules than eosin Y-conjugated streptavidin (Figure S7). The unexpectedly low signal enhancement is likely due to slow release kinetics of photoactive, monomeric eosin Y from liposomes in 1×PBST compared to the time scale of PBA (~100 seconds) (Figure S8, Figure S9). On the basis of the 30-fold enhancement in sensitivity, one might think that we could also achieve 30-fold improvement in detection limit with liposome-enhanced PBA. However, this was not observed. As shown in Figure 2B, signal-to-noise ratio of MFI in the liposome assay exceeded two at 3 nM of streptavidin, but the conventional assay required 10 nM of streptavidin to reach the same level, which may imply 3-fold improvement in detection limit of PBA when using liposomes. The main reason for this lesser improvement in detection limit is that the liposome assay produced relatively high non-specific binding signals (MFI = 469) compared to very small non-specific binding signals (MFI = 69) in the conventional assay. We suggest this higher noise in the liposome assay could make it much more difficult to achieve signal-to-noise ratio of two than in the conventional assay.


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Figure 4. Quantification of liposome-enhanced PBA and conventional PBA results. Paper-based liposome and conventional assays were performed with a dilution series (0 nM to 300 nM) of streptavidin and eosin Y-conjugated streptavidin (SA-E) respectively, followed by PBA with monomer solution* as described in Scheme 1. (A) Representative images of PBA results. The test zones were imaged immediately after the addition of 2 μL of 0.5 M NaOH solution. Liposomeenhanced PBA still required alkaline environment to visualize hydrogels as the amount of eosin Y released from liposomes was not enough to stain the hydrogels (Figure S10). Replicates are shown in Figure S11. (B) Colorimetric intensity (ΔCIE) values were calculated using the method previously described.17 Limit of detection (LOD) was determined as the minimum concentration of streptavidin or eosin Y-conjugated streptavidin at which all replicates give a ΔCIE value that is larger than the ΔCIE value from the negative controls by at least three standard deviations of the


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negative controls. Each data point is an average of three replicates and the error bars represent standard deviations. *The monomer solution included 200 mM poly(ethylene glycol) diacrylate (PEGDA), 150 mM triethanolamine (TEOA), 100 mM 1-vinyl-2-pyrrolidinone (VP), 20 mM hydrochloric acid (HCl), 1.6 mM phenolphthalein, and 0.4 μM eosin Y in deionized water.

Comparison of liposome-enhanced PBA and conventional PBA Finally, we assessed whether liposome-enhanced PBA would produce a larger amount of colored hydrogel than conventional PBA for the same analyte concentration. As presented in Figure 4, liposome-enhanced PBA reached the maximum colorimetric signal even at 10 nM of streptavidin while conventional PBA could achieve similar colorimetric intensity at 300 nM of streptavidin. Furthermore, liposome-enhanced PBA with 3 nM of streptavidin produced a similar amount of polymer film to that obtained with conventional PBA with 100 nM of streptavidin. These observations indicate 30-fold enhancement in sensitivity as we obtained from the analysis of background-subtracted MFI. Although one liposome rapidly released about 11,000 free eosin Y molecules in monomer solution, the sensitivity of PBA was not improved as much as this number would suggest (Figure S9). The presence of non-specifically bound liposomes, which could release the same number of free eosin Y molecules, prevented realization of more dramatic improvements in sensitivity. The limit of detection of liposome-enhanced PBA was 3 nM, and the limit of detection of conventional PBA was 10 nM as estimated by the analysis of signal-to-noise ratio of MFI. This result also supports our observation that signal-to-noise ratio of MFI should be at least two to obtain colorimetric signals.17,23 From these results, we confirmed that liposome-


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enhanced PBA could amplify specific binding signals more efficiently than conventional PBA due to faster polymerization with greater amount of free eosin Y localized to binding region. It is interesting to note that the dynamic range of liposome-enhanced PBA is much narrower than that of conventional PBA. It is likely because PBA has an intrinsic maximum colorimetric intensity and the liposome-enhanced PBA reaches the maximum at lower analyte concentration due to its higher sensitivity. With our cell phone-based imaging method, we could not capture the actual amount of polymer once polymer film covered the whole areas of test zones. As a result, there was no difference in colorimetric signals with more than 10 nM of the analyte in liposomeenhanced PBA. The narrow dynamic range can be useful for rapid diagnostic tests3,8,47 where one only needs to detect analytes qualitatively. Given that untrained users might get confused with very small colorimetric signals such as those provided by conventional PBA at 3 nM to 30 nM of the analyte, the high sensitivity of liposome-enhanced PBA may lower the risk of subjective interpretation of test results. For other applications48–50 such as monitoring disease progression and treatment efficacy that require quantitative analysis over extended dynamic range, liposomeenhanced PBA may not be practical because of its narrow dynamic range. However, the dynamic range in PBA is not intrinsically determined, and it can be extended by using shorter irradiation time,17 lowering the surface density of capture proteins,51 and combining multiple binders with different binding affinity for the same target.52


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Figure 5. Quantification of colorimetric intensity of liposome-enhanced PBA with 10×volume of analyte solution. Paper-based liposome assays were performed with 100 μL of a dilution series (0 nM to 300 nM) of streptavidin, followed by PBA as described in Scheme 1. (A) Representative images of PBA results. The test zones were imaged immediately after the addition of 2 μL of 0.5 M NaOH solution. (B) Colorimetric intensity (ΔCIE) values were calculated using the method previously described.17 Limit of detection (LOD) was determined as the minimum concentration of streptavidin or eosin Y-conjugated streptavidin at which all replicates give a ΔCIE value that is larger than the ΔCIE value from the negative controls by at least three standard deviations of the negative controls. Each data point is an average of three replicates and the error bars are standard deviations.


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Liposome-enhanced PBA with 10×volume of analyte solution We also investigated whether large volume of analyte solution can further improve detection limit of liposome-enhanced PBA. As we discussed above, incorporation of liposomes could not improve detection limit as much as the improvement in sensitivity because of relatively large nonspecific binding signals at low analyte concentrations. Considering this result, we decided to increase the volume of the analyte solution in our flow-through assays because it could only increase specific binding signals without accompanying increase in non-specific binding signals.53 Instead of incubating 10 μL of analyte solution with test zones, we continuously added 100 μL of analyte solution on test zones over 30 minutes. This flow rate could ensure that the rate of analyte capture was larger than the rate of convective mass transport in the binding region (Figure S12). Figure 5 demonstrated that 10×volume of analyte solution successfully improved the detection limit by ten-fold (3 nM to 0.3 nM). The signal-to-noise ratio of MFI first exceeded two at 0.3 nM, which is consistent with our previous observation (Figure S13). Sensitivity also increased approximately by ten-fold compared to that of the small volume (10 μL) case (Figure S14). It should be of note that the same extent of improvements in detection limit and sensitivity were obtained in the flow-through assay, which is different from earlier comparison between liposomeenhanced PBA and conventional PBA. We suggest that it is mainly because the flow-through assay selectively increased specific binding signals with larger amount of total analytes without changing non-specific binding signals (Figure S15). This finding may support the possibility that liposomeenhanced PBA can sensitively detect picomolar concentration of analytes with larger volume (1 mL to 10 mL) of sample in flow-through assays. If this is the case, we envision that liposomeenhanced PBA will also be suited for paper-based diagnostic tests where high sensitivity is required and large volume of sample is available such as urine-based tests.54–57


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CONCLUSIONS

In summary, we demonstrated here a highly sensitive colorimetric paper-based biodetection method incorporating liposomes into polymerization-based signal amplification (PBA). Liposomes were developed to localize a greater amount of photoactive eosin Y to specifically captured protein and amplify the binding signals more efficiently than conventional PBA, where a few conjugated eosin Y molecules per binding event initiate polymerization. Fluorescence microscopy results implied that liposome-enhanced PBA achieved 30-fold improvement in sensitivity and 3-fold improvement in detection limit compared to conventional PBA. These predictions were confirmed by colorimetric PBA results. Relatively high non-specific binding signals in liposome-enhanced PBA compared to conventional PBA prevented further improvements. Thus, 10×volume of analyte solution was used in continuous flow-through assays to selectively increase specific binding signals while maintaining non-specific binding signals. Using this approach, both sensitivity and detection limit of liposome-enhanced PBA were further improved by ten-fold, achieving sub-nanomolar naked-eye biodetection. Designing eosin Ycarriers featuring reduced non-specific binding to biofunctionalized paper is a promising future direction that may further improve the detection limit of PBA from the sub-nanomolar to the picomolar range, increasing applicability for early diagnosis of many diseases.24

EXPERIMENTAL SECTION

Materials


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Phospholipids for liposome synthesis including 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG(2000), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG(2000)-Biotin) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol (≥99%), chloroform (anhydrous, ≥99%), methanol (HPLC Plus, ≥99.9%), phenolphthalein (≥98%), poly(ethylene glycol) diacrylate (PEGDA, average Mn = 575), triethanolamine

(TEOA,

98%),

1-vinyl-2-pyrrolidinone

(VP,

>99%),

2’,4’,5’,7’-

tetrabromofluorescein disodium salt (eosin Y, ≥85%), dimethyl sulfoxide (DMSO, ≥99%), 10×phosphate buffered saline (PBS), Tween® 20, and Sephadex® G-50 (Fine, GE Healthcare) were purchased from Sigma Aldrich (St. Louis, MO, USA). Hydrochloric acid (HCl), sodium hydroxide (NaOH), Whatman No. 1 chromatography paper, and glycerol were purchased from VWR (Radnor, PA, USA). Bovine serum albumin (10% w/v) in 1×PBS was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Eosin 5-isothiocyanate (EITC, ≥98%) was purchased from Marker Gene Technology (Eugene, OR, USA). Streptavidin was obtained from Rockland Immunochemicals Inc. (Limerick, PA, USA). CBD-fused, streptavidin binding variant of reduced charge protein Sso7d from Sulfolobus solfataricus was expressed and purified by Eric A. Miller as described in his paper.45 All reagents were used as received without further purification.

Liposome synthesis Liposomes were synthesized using the thin film hydration method,38 and disodium salt form of eosin Y was actively loaded using transmembrane calcium acetate gradients.36 Lipid formulation of DPPC : DPPG : cholesterol : DSPE-PEG(2000) : DSPE-PEG(2000)-Biotin = 34 : 21.5 : 43 : 1


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: 0.5 was determined by consulting literature40,58 in terms of colloidal stability, non-specific binding, and dye loading efficiency. To prepare 3 mL of 1.26 mg/mL liposomes, 3.8 mg of the phospholipids and cholesterol with the desired molar ratio were dissolved in 1 mL of chloroform : methanol = 3 : 1 solution. Organic solvent was evaporated using rotary evaporator, and the lipid film was dried under vacuum for at least an hour. The dried lipid film was rehydrated by adding 1 mL of 100 mM calcium acetate (pH 4.2). The lipid solution was vigorously agitated, shaken, and stirred for an hour at 50°C. The resulting turbid solution was passed 15 times at 70°C through a polycarbonate membrane with a pore diameter of 400 nm. Depending on the desired size of liposomes, the extruded liposomes were passed 15 times more at 70°C through a polycarbonate membrane with a pore diameter of 50 nm. To eliminate unentrapped calcium acetate, 1 mL of the extruded liposome solution was dialyzed against 1 L of 100 mM sodium sulfate (pH 4.2) overnight in cold room (4°C). To the dialyzed liposome solution, eosin Y disodium salt was added with molar ratios (0.125 or 1.25) of the dye to phospholipids and cholesterol. After incubating the solution at 50°C for two hours, unentrapped eosin Y was separated from eosin Y-loaded liposomes by size exclusion chromatography using Sephadex® G-50 as the stationary phase and 1 × PBS as the mobile phase. The eluted liposomes were diluted to a final lipid concentration of 1.26 mg/mL and stored at 4°C until use.

Liposome characterization The size and % polydispersity of liposomes were determined by dynamic light scattering (DLS) using DynaPro NanoStar (Wyatt Technology, Santa Barbara, CA, USA). Liposome solution was diluted 3-fold prior to this DLS measurement. The amount of unentrapped eosin Y was quantified by UV-vis spectroscopy (λ = 522 nm), and eosin Y-loading efficiency was calculated by the


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following equation: (mol of total eosin Y used in the loading step − mol of unentrapped eosin Y) ÷ (mol of phospholipids in liposome solution). Stability of the optimized liposome at 4°C was monitored using its performance in streptavidin assays, indicating that the liposome was stable at 4°C for at least a month (Figure S16).

Paper-based streptavidin detection assays Paper-based flow-through assays for streptavidin were conducted using the method previously described17 with a slight modification. Test strips were made by printing a wax-mask containing circular wax-free region on Whatman No. 1 chromatography paper and melting the paper in an oven (150°C) for 90 seconds. The wax-free test zones were rinsed with deionized water, and the washed zones were placed in a humid chamber in a way that only edge of test strips were contacted with other surfaces. Then, 6 μL of rcSso7d.SA-CBD solution (30 μM in 1×PBS) was added to each test zone and incubated with the cellulose surface for ten minutes. Unimmobilized rcSso7d.SA-CBD was washed with 1×PBS, and each test zone was contacted with 10 μL of a specific concentration of streptavidin or eosin Y-conjugated streptavidin (0 nM to 300 nM) in 1% PBSA (1% BSA in 1×PBS) for 30 minutes in the humid chamber. For eosin Y-conjugated streptavidin assays, each test zone was washed with 1×PBS, 1×PBST (0.1% Tween-20 in 1×PBS), and deionized water, followed by drying. For the optimized liposome-based streptavidin assays, each streptavidin-captured test zone was washed with 1×PBS, and the immobilized streptavidin was contacted with 10 μL of biotinylated eosin Y-loaded liposomes (0.18 mM lipids) in 1% PBSA for 30 minutes. The unbound liposomes were removed by washing the test zones three times with 1×PBS and two times with 1×PBST, and the test zones were allowed to dry completely. All


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washing steps included adding 20 μL of washing buffer or deionized water on a test zone and letting it flow through the hydrophilic test zone.

Fluorescence microscopy Each test zone was imaged using an Olympus IX81 microscope with a Semrock TxRed-4040C filter set. The exposure time of 500 ms or 100 ms was used depending on fluorescence intensity of each test zone to prevent detector saturation. Using linear relationship between exposure time and fluorescence intensity (Figure S17), the fluorescence intensity measured with 100 ms of exposure time was multiplied by five to convert it to the fluorescence intensity that would be obtained with 500 ms of exposure time. The mean fluorescence intensity of each test zone was calculated by averaging the pixel intensities using ImageJ as previously described.59

Polymerization-based signal amplification (PBA) After running paper-based assays, each dry test zone was washed with 20 μL of 1×PBS and contacted with 10 μL of monomer solution containing 200 mM PEGDA, 100 mM VP, 150 mM TEOA, 0.4 μM eosin Y, 1.6 mM phenolphthalein, and 20 mM HCl. The surface was illuminated with green light from an ampliPHOX (λmax = 522 nm, FWHM = 30 nm, 25 mW/cm2) for 55 seconds (liposome-enhanced PBA with 10×volume of analyte solution), 70 seconds (liposomeenhanced PBA), or 100 seconds (conventional PBA), each of which is the longest time that cannot initiate bulk polymerization with negative control samples. The unpolymerized monomer solution was rinsed with a rinsing bottle by washing each surface of the test zone for 10 seconds. The test zone was placed on blotting paper and rinsed twice with 20 μL of deionized water. To visualize


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the test result, 2 μL of 0.5 M NaOH solution was added to the test zone. All surfaces were imaged immediately after the addition of the basic solution.

Imaging PBA results PBA results were imaged with a smartphone, Samsung Galaxy S8, using its camera in the HDR mode. To control lighting conditions, the phone was fixed on a ring stand at the same position, and an ordinary desk lamp was used to illuminate via diffuse reflection the test surface that was placed under the phone. The images were used without modification.

ASSOCIATED CONTENT

Supporting Information. Experimental details, supplementary data for optimization and assessment of liposome-enhanced polymerization-based signal amplification (PBA), solution phase eosin Y release kinetics, fluorescence imaging results of liposome-enhanced PBA with 10×volume of analyte solution, and stability of liposomes. This material is available for free of charge on ACS Publications website.

AUTHOR INFORMATION

Corresponding Author *E-mail for H.D.S.: [email protected].


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ORCID Seunghyeon Kim: 0000-0001-6515-2679 Hadley D. Sikes: 0000-0002-7096-138X Notes The authors declare that they have no competing interests. Author Contributions The manuscript was written through contributions of both authors. Both authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENTS

This research was supported by the Singapore National Research Foundation under its Antimicrobial Resistance IRG administered by the Singapore Alliance for Research and Technology. HDS also acknowledges support from an Esther and Harold E. Edgerton professorship. SK acknowledges support from the Kwanjeong Educational Foundation. The authors thank Eric A. Miller for expression and purification of rcSso7d.SA-CBD.

REFERENCES

(1)

Parolo, C.; Merkoçi, A. Paper-Based Nanobiosensors for Diagnostics. Chem. Soc. Rev. 2013, 42 (2), 450–457.


24

Page 25 of 33 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


(2)

Hu, J.; Wang, S.; Wang, L.; Li, F.; Pingguan-Murphy, B.; Lu, T. J.; Xu, F. Advances in Paper-Based Point-of-Care Diagnostics. Biosens. Bioelectron. 2014, 54, 585–597.

(3)

Mukkala, A. N.; Kwan, J.; Lau, R.; Harris, D.; Kain, D.; Boggild, A. K. An Update on Malaria Rapid Diagnostic Tests. Curr. Infect. Dis. Rep. 2018, 20 (12), 49, 1–8.

(4)

Figueroa, C.; Johnson, C.; Ford, N.; Sands, A.; Dalal, S.; Meurant, R.; Prat, I.; Hatzold, K.; Urassa, W.; Baggaley, R. Reliability of HIV Rapid Diagnostic Tests for Self-Testing Compared with Testing by Health-Care Workers: A Systematic Review and Meta-Analysis. Lancet HIV 2018, 5 (6), e277–e290.

(5)

Gubala, V.; Harris, L. F.; Ricco, A. J.; Tan, M. X.; Williams, D. E. Point of Care Diagnostics: Status and Future. Anal. Chem. 2012, 84 (2), 487–515.

(6)

Yetisen, A. K.; Akram, M. S.; Lowe, C. R. Paper-Based Microfluidic Point-Of-Care Diagnostic Devices. Lab Chip 2013, 13 (12), 2210–2251.

(7)

Bahadır, E. B.; Sezgintürk, M. K. Lateral Flow Assays: Principles, Designs and Labels. Trends Anal. Chem. 2016, 82, 286–306.

(8)

Cunningham, J.; Hasker, E.; Das, P.; Safi, S. El; Goto, H.; Mondal, D.; Mbuchi, M.; Mukhtar, M.; Rabello, A.; Rijal, S.; Sundar, S.; Wasunna, M.; Adams, E.; Menten, J.; Peeling, R.; Boelaert, M. A Global Comparative Evaluation of Commercial Immunochromatographic Rapid Diagnostic Tests for Visceral Leishmaniasis. Clin. Infect. Dis. 2012, 55 (10), 1312–1319.

(9)

Barbosa, A. I.; Wichers, J. H.; Amerongen, A. van; Reis, N. M. Towards One-Step Quantitation of Prostate-Specific Antigen (PSA) in Microfluidic Devices: Feasibility of


25


Page 26 of 33

Optical Detection with Nanoparticle Labels. Bionanoscience 2017, 7 (4), 718–726. (10)

Mao, X.; Ma, Y.; Zhang, A.; Zhang, L.; Zeng, L.; Liu, G. Disposable Nucleic Acid Biosensors Based on Gold Nanoparticle Probes and Lateral Flow Strip. Anal. Chem. 2009, 81 (4), 1660–1668.

(11)

Jiang, T.; Song, Y.; Wei, T.; Li, H.; Du, D.; Zhu, M. J.; Lin, Y. Sensitive Detection of Escherichia Coli O157:H7 Using Pt-Au Bimetal Nanoparticles with Peroxidase-Like Amplification. Biosens. Bioelectron. 2016, 77, 687–694.

(12)

Loynachan, C. N.; Thomas, M. R.; Gray, E. R.; Richards, D. A.; Kim, J.; Miller, B. S.; Brookes, J. C.; Agarwal, S.; Chudasama, V.; McKendry, R. A.; Stevens, M. M. Platinum Nanocatalyst Amplification: Redefining the Gold Standard for Lateral Flow Immunoassays with Ultrabroad Dynamic Range. ACS Nano 2018, 12 (1), 279–288.

(13)

Ye, H.; Xia, X. Enhancing the Sensitivity of Colorimetric Lateral Flow Assay (CLFA) through Signal Amplification Techniques. J. Mater. Chem. B 2018, 6 (44), 7102–7111.

(14)

Zhang, J.; Gui, X.; Zheng, Q.; Chen, Y.; Ge, S.; Zhang, J.; Xia, N. An HRP-Labeled Lateral Flow Immunoassay for Rapid Simultaneous Detection and Differentiation of Influenza A and B Viruses. J. Med. Virol. 2019, 91 (3), 503–507.

(15)

Lathwal, S.; Sikes, H. D. Assessment of Colorimetric Amplification Methods in a PaperBased Immunoassay for Diagnosis of Malaria. Lab Chip 2016, 16 (8), 1374–1382.

(16)

Watson, V.; Dacombe, R. J.; Williams, C.; Edwards, T.; Adams, E. R.; Johnson, C.; Mutseta, M. N.; Corbett, E. L.; Cowan, F. M.; Ayles, H.; Hatzold, K.; MacPherson, P.; Taegtmeyer, M. Re-Reading of OraQuick HIV-1/2 Rapid Antibody Test Results: Quality


26

Page 27 of 33 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


Assurance Implications for HIV Self-Testing Programmes. J. Int. AIDS Soc. 2019, 22 (S1), e25234, 19–24. (17)

Badu-Tawiah, A. K.; Lathwal, S.; Kaastrup, K.; Al-Sayah, M.; Christodouleas, D. C.; Smith, B. S.; Whitesides, G. M.; Sikes, H. D. Polymerization-Based Signal Amplification for Paper-Based Immunoassays. Lab Chip 2015, 15 (3), 655–659.

(18)

Aguirre-Soto, A.; Kaastrup, K.; Kim, S.; Ugo-Beke, K.; Sikes, H. D. Excitation of Metastable Intermediates in Organic Photoredox Catalysis: Z-Scheme Approach Decreases Catalyst Inactivation. ACS Catal. 2018, 8 (7), 6394–6400.

(19)

Aguirre-Soto, A.; Kim, S.; Kaastrup, K.; Sikes, H. D. On the Role of N-Vinylpyrrolidone in the Aqueous Radical-Initiated Copolymerization with PEGDA Mediated by Eosin Y in the Presence of O2. Polym. Chem. 2019, 10 (8), 926–937.

(20)

Kaastrup, K.; Sikes, H. D. Using Photo-Initiated Polymerization Reactions to Detect Molecular Recognition. Chem. Soc. Rev. 2016, 45 (3), 532–545.

(21)

Dellal, D.; Yee, E.; Lathwal, S.; Sikes, H.; Gomez-Marquez, J. Low-Cost Plug and Play Photochemistry Reactor. HardwareX 2018, 3, 1–9.

(22)

Kim, S.; Sikes, H. D. Phenolphthalein-Conjugated Hydrogel Formation under Visible-Light Irradiation for Reducing Variability of Colorimetric Biodetection. ACS Appl. Bio Mater. 2018, 1 (2), 216–220.

(23)

Yee, E. H.; Lathwal, S.; Shah, P. P.; Sikes, H. D. Detection of Biomarkers of Periodontal Disease in Human Saliva Using Stabilized, Vertical Flow Immunoassays. ACS Sensors 2017, 2 (11), 1589–1593.


27


(24)

Page 28 of 33

Kelley, S. O. What Are Clinically Relevant Levels of Cellular and Biomolecular Analytes? ACS Sensors 2017, 2 (2), 193–197.

(25)

Lee, J. K.; Sikes, H. D. Balancing the Initiation and Molecular Recognition Capabilities of Eosin Macroinitiators of Polymerization-Based Signal Amplification Reactions. Macromol. Rapid Commun. 2014, 35 (10), 981–986.

(26)

Kaastrup, K.; Sikes, H. D. Investigation of Dendrimers Functionalized with Eosin as Macrophotoinitiators for Polymerization-Based Signal Amplification Reactions. RSC Adv. 2015, 5 (20), 15652–15659.

(27)

Qian, H.; He, L. Polymeric Macroinitiators for Signal Amplification in AGET ATRP-Based DNA Detection. Sensors Actuators B 2010, 150 (2), 594–600.

(28)

Cui, Y.; Zhuang, D.; Tan, T.; Yang, J. Highly Sensitive Visual Detection of Mutant DNA Based on Polymeric Nanoparticles-Participating Amplification. RSC Adv. 2016, 6 (116), 115238–115246.

(29)

Sikes, H. D.; Hansen, R. R.; Johnson, L. M.; Jenison, R.; Birks, J. W.; Rowlen, K. L.; Bowman, C. N. Using Polymeric Materials to Generate an Amplified Response to Molecular Recognition Events. Nat. Mater. 2008, 7 (1), 52–56.

(30)

Lee, J. K.; Heimer, B. W.; Sikes, H. D. Systematic Study of Fluorescein-Functionalized Macrophotoinitiators for Colorimetric Bioassays. Biomacromolecules 2012, 13 (4), 1136– 1143.

(31)

Enoki, M.; Katoh, R. Estimation of Quantum Yields of Weak Fluorescence from Eosin Y Dimers Formed in Aqueous Solutions. Photochem. Photobiol. Sci. 2018, 17 (6), 793–799.


28

Page 29 of 33 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


(32)

Bae, Y. H.; Park, K. Targeted Drug Delivery to Tumors: Myths, Reality and Possibility. J. Control. Release 2011, 153 (3), 198–205.

(33)

Allen, T. M.; Cullis, P. R. Liposomal Drug Delivery Systems: From Concept to Clinical Applications. Adv. Drug Deliv. Rev. 2013, 65 (1), 36–48.

(34)

Yingchoncharoen, P.; Kalinowski, D. S.; Richardson, D. R. Lipid-Based Drug Delivery Systems in Cancer Therapy: What Is Available and What Is Yet to Come. Pharmacol. Rev. 2016, 68 (3), 701–787.

(35)

Haran, G.; Cohen, R.; Bar, L. K.; Barenholz, Y. Transmembrane Ammonium Sulfate Gradients in Liposomes Produce Efficient and Stable Entrapment of Amphipathic Weak Bases. Biochim. Biophys. Acta 1993, 1151 (2), 201–215.

(36)

Clerc, S.; Barenholz, Y. Loading of Amphipathic Weak Acids into Liposomes in Response to Transmembrane Calcium Acetate Gradients. Biochim. Biophys. Acta 1995, 1240 (2), 257–265.

(37)

Fritze, A.; Hens, F.; Kimpfler, A.; Schubert, R.; Peschka-Süss, R. Remote Loading of Doxorubicin into Liposomes Driven by a Transmembrane Phosphate Gradient. Biochim. Biophys. Acta 2006, 1758 (10), 1633–1640.

(38)

Zhang, H. Thin-Film Hydration Followed by Extrusion Method for Liposome Preparation. In Liposomes: Methods and Protocols, Methods in Molecular Biology; 2017; Vol. 1522, pp 17–22.

(39)

Sawant, R. R.; Torchilin, V. P. Challenges in Development of Targeted Liposomal Therapeutics. AAPS J. 2012, 14 (2), 303–315.


29


(40)

Page 30 of 33

Edwards, K. A.; Meyers, K. J.; Leonard, B.; Connelly, J. T.; Wang, Y.; Holter, T.; Baeumner, A. J. Engineering Liposomes as Detection Reagents for CD4+ T-Cells. Anal. Methods 2012, 4 (12), 3948–3955.

(41)

Wen, H. W.; Borejsza-Wysocki, W.; Decory, T. R.; Durst, R. A. Development of a Competitive Liposome-Based Lateral Flow Assay for the Rapid Detection of the Allergenic Peanut Protein Ara H1. Anal. Bioanal. Chem. 2005, 382 (5), 1217–1226.

(42)

Edwards, K. A.; Baeumner, A. J. Liposome-Enhanced Lateral-Flow Assays for the Sandwich-Hybridization Detection of RNA. In Methods in Molecular Biology: Biosensors and Biodetection; 2009; Vol. 504, pp 185–215.

(43)

Edwards, K. A.; Korff, R.; Baeumner, A. J. Liposome-Enhanced Lateral-Flow Assays for Clinical Analyses. In Biosensors and Biodetection: Methods and Protocols Volume 1: Optical-based detectors, Methods in Molecular Biology; 2017; Vol. 1571, pp 407–434.

(44)

Frohnmeyer, E.; Tuschel, N.; Sitz, T.; Hermann, C.; Dahl, G. T.; Schulz, F.; Baeumner, A. J.; Fischer, M. Aptamer Lateral Flow Assays for Rapid and Sensitive Detection of Cholera Toxin. Analyst 2019, 144 (5), 1840–1849.

(45)

Miller, E. A.; Baniya, S.; Osorio, D.; Maalouf, Y. J. Al; Sikes, H. D. Paper-Based Diagnostics in the Antigen-Depletion Regime: High-Density Immobilization of RcSso7dCellulose-Binding Domain Fusion Proteins for Efficient Target Capture. Biosens. Bioelectron. 2018, 102, 456–463.

(46)

Wu, Y.; Tilley, R. D.; Gooding, J. J. Challenges and Solutions in Developing Ultrasensitive Biosensors. J. Am. Chem. Soc. 2019, 141 (3), 1162–1170.


30

Page 31 of 33 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


(47)

Barbosa, J. R.; Colares, J. K. B.; Flores, G. L.; Cortes, V. F.; Miguel, J. C.; Portilho, M. M.; Marques, V. A.; Potsch, D. V.; Brandão-Mello, C. E.; Amendola-Pires, M.; Pilotto, J. H.; Lima, D. M.; Lampe, E.; Villar, L. M. Performance of Rapid Diagnostic Tests for Detection of Hepatitis B and C Markers in HIV Infected Patients. J. Virol. Methods 2017, 248, 244– 249.

(48)

Langford, S. E.; Ananworanich, J.; Cooper, D. A. Predictors of Disease Progression in HIV Infection: A Review. AIDS Res. Ther. 2007, 4 (11), 1–14.

(49)

Shayo, A.; Buza, J.; Ishengoma, D. S. Monitoring of Efficacy and Safety of ArtemisininBased Anti-Malarials for Treatment of Uncomplicated Malaria: A Review of Evidence of Implementation of Anti-Malarial Therapeutic Efficacy Trials in Tanzania. Malar. J. 2015, 14 (1), 135, 1–12.

(50)

Pena, M. J.; Stenvinkel, P.; Kretzler, M.; Adu, D.; Agarwal, S. K.; Coresh, J.; Feldman, H. I.; Fogo, A. B.; Gansevoort, R. T.; Harris, D. C.; Jha, V.; Liu, Z.; Luyckx, V. A.; Massy, Z. A.; Mehta, R.; Nelson, R. G.; O’Donoghue, D. J.; Obrador, G. T.; Roberts, C. J.; Sola, L.; Sumaili, E. K.; Tatiyanupanwong, S.; Thomas, B.; Wiecek, A.; Parikh, C. R.; Heerspink, H. J. L. Strategies to Improve Monitoring Disease Progression, Assessing Cardiovascular Risk, and Defining Prognostic Biomarkers in Chronic Kidney Disease. Kidney Int. Suppl. 2017, 7 (2), 107–113.

(51)

Lathwal, S.; Sikes, H. D. A Method for Designing Instrument-Free Quantitative Immunoassays. Anal. Chem. 2016, 88 (6), 3194–3202.

(52)

Vallée-Bélisle, A.; Ricci, F.; Plaxco, K. W. Engineering Biosensors with Extended, Narrowed, or Arbitrarily Edited Dynamic Range. J. Am. Chem. Soc. 2012, 134 (6), 2876–


31


Page 32 of 33

2879. (53)

Miller, E. A.; Maalouf, Y. J. Al; Sikes, H. D. Design Principles for Enhancing Sensitivity in Paper-Based Diagnostics via Large-Volume Processing. Anal. Chem. 2018, 90 (15), 9472–9479.

(54)

Thomas, C. E.; Sexton, W.; Benson, K.; Sutphen, R.; Koomen, J. Urine Collection and Processing for Protein Biomarker Discovery and Quantification. Cancer Epidemiol. Biomarkers Prev. 2010, 19 (4), 953–959.

(55)

Pollock, N. R.; Macovei, L.; Kanunfre, K.; Dhiman, R.; Restrepo, B. I.; Zarate, I.; Pino, P. a; Mora-Guzman, F.; Fujiwara, R. T.; Michel, G.; Kashino, S. S.; Campos-Neto, A. Validation of Mycobacterium Tuberculosis Rv1681 Protein as a Diagnostic Marker of Active Pulmonary Tuberculosis. J. Clin. Microbiol. 2013, 51 (5), 1367–1373.

(56)

An, M.; Gao, Y. Urinary Biomarkers of Brain Diseases. Genomics Proteomics Bioinforma. 2015, 13 (6), 345–354.

(57)

Paris, L.; Magni, R.; Zaidi, F.; Araujo, R.; Saini, N.; Harpole, M.; Coronel, J.; Kirwan, D. E.; Steinberg, H.; Gilman, R. H.; Petricoin, E. F.; Nisini, R.; Luchini, A.; Liotta, L. Urine Lipoarabinomannan Glycan in HIV-Negative Patients with Pulmonary Tuberculosis Correlates with Disease Severity. Sci. Transl. Med. 2017, 9, 1–11.

(58)

Edwards, K. A.; Meyers, K. J.; Leonard, B.; Baeumner, A. J. Superior Performance of Liposomes Over Enzymatic Amplification in a High-Throughput Assay for Myoglobin in Human Serum. Anal. Bioanal. Chem. 2013, 405 (12), 4017–4026.


32

Page 33 of 33 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


(59)

Miller, E. A.; Traxlmayr, M. W.; Shen, J.; Sikes, H. D. Activity-Based Assessment of an Engineered Hyperthermophilic Protein as a Capture Agent in Paper-Based Diagnostic Tests. Mol. Syst. Des. Eng. 2016, 1 (4), 377–381.

TABLE OF CONTENTS (TOC) / ABSTRACT GRAPHIC


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Liposome-Enhanced Polymerization-Based Signal Amplification for

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