Nanoscale probes for the evaluation of the integrity of ultrafiltration membranes

Journal of Membrane Science 276 (2006) 199–207

Nanoscale probes for the evaluation of the integrityof ultrafiltration membranes

Vitaly Gitis a,∗, Roy C. Haught b, Robert M. Clark c,Jenny Gun d, Ovadia Lev d

a Department of Biotechnology and Environmental Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israelb Water Quality Management Branch, Water Supply and Water Resources Division, National Risk Management Research Laboratory,

Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH 45268, USAc Environmental Engineer and Public Health Consultant, 9627 Lansford Cr., Cincinnati, OH 45242, USA

d Laboratory of Environmental Chemistry, Faculty of Science, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Received 14 July 2005; received in revised form 21 September 2005; accepted 22 September 2005Available online 17 November 2005

Abstract

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Current integrity tests are not sufficiently sensitive to detect nanometric scale breaches in the active layer of ultrafiltration membranes. The currentaper describes a new approach for the detection of such breaches. We introduce two representative types of nanoprobes emulating virus transportver the membrane. Gold nanoparticles and fluorescent-dye-labeled MS2 bacteriophages were introduced for seeding tests. The affordability of theormer probe has become more realistic with the development of electrochemical detection of gold nanoparticles by anodic stripping voltammetryASV). Our ASV system showed high sensitivity, of the order of single parts per billion, indicating the feasibility of developing the experimentalrotocol for on-site analysis. However, the physical characteristics of gold nanoparticles and particularly their specific density differ from thosef viruses. The fluorescent bacteriophage probe emulates viral transport much better, though this technique is less sensitive, and further loweringf the limit of detection is required. Application of the probes for testing membrane integrity will provide a basis for developing on-line testingor removal of virus-sized particles and in addition to being a valuable research tool may provide a means to confirm compliance of membraneystems with the stringent regulatory requirements of the drinking water industry.

2005 Elsevier B.V. All rights reserved.

eywords: Citrate-stabilized gold nanoparticles; MS2 bacteriophages; Integrity breaches; Nanoprobes

. Introduction

Membrane systems are being increasingly used for the directreatment of drinking water. Over 300 large water treatmentacilities located in the USA and Europe are currently based onicrofiltration (MF) and ultrafiltration (UF) membrane mod-

les. However, the lack of means to accurately monitor mem-rane integrity over long-term operation is preventing the fullxploitation of the potential of UF technologies for water treat-ent. Membrane integrity, i.e., the absence of feed leakage

hrough affected or broken membranes, or passage through seals,an be compromised as a result of factory defects, improper

∗ Corresponding author. Tel.: +972 86479031.E-mail address: [email protected] (V. Gitis).

shipping and maintenance, faulty integrity tests, and excessiveexposure to chemicals during chemical clean-up.

Four direct and three indirect integrity tests for detection ofsystem breaches are currently in use [1–3]. The direct integritytesting methods – acoustic noise analysis, the bubble point test,the pressure decay test (PDT), and the diffusive air flow (DAF)test – are not suitable for on-line site testing for a number ofreasons, one being that they are not sufficiently sensitive. Theminimal detectable level for the three pressure-integrity tests(i.e., bubble point test, PDT, and DAF test) is 2–3 �m, a detectionlimit that is sufficient to assure compliance with removal require-ments for Giardia (∼6–20 �m), Cryptosporidium (∼4–6 �m),and other parasites but not viruses (�0.1 �m). In addition tothe drawback of their limited sensitivity for large plants [4], thePDT and the DAF test may at times cause membrane breakageduring the test and may yield false-positive results due to the

376-7388/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.memsci.2005.09.048

200 V. Gitis et al. / Journal of Membrane Science 276 (2006) 199–207

membrane being partially unwetted (after backwash) or due toseasonal viscosity changes [1]. These tests also suffer from sub-jective interpretation of pressure deterioration. Thus, plants thatrely on pressure-integrity tests alone are likely to continue theiroperation beyond the point where their membrane integrity hasbeen compromised. The absence of a reliable sensitive on-linedetection method for monitoring of membrane integrity is cur-rently hampering the allocation of more significant virus removalcredits for UF membranes.

Indirect integrity tests include particle counting, turbiditymonitoring, and surrogate challenge tests. In all these tests,changes in filtrate quality are monitored by comparing test probelevels with a previously established baseline level. These testsare very convenient for routine qualitative monitoring, but theirinterpretation in terms of pathogen permeability is questionable,and they are less likely to meet the quality control requirementsof regulatory agencies. The tests are not sufficiently sensitive todetect individual fibre pinholes that result from challenge operat-ing conditions [2]. Additionally, the indirect tests do not providea means to identify the location of membrane breaches and mayinvolve a significant increase in capital costs for particle coun-ters. Recently, van Hoof et al. [5] proposed an on-line surrogatechallenge test using a spike injection or a constant feed of pow-dered activated carbon (PAC), followed by optical monitoringof filtrate quality with particle or turbidity monitors. This test is,however, not suitable from a regulatory point of view, since thetPtow

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ing of fluorescent-dye-labeled bacteriophages has been reportedpreviously [7]. The quantification of gold nanoparticles in watersamples did, however, require development work, and some ofthe technical difficulties are discussed below.

To be implemented for routine monitoring of colloidal aque-ous dispersions, the gold detection method should combineaccuracy, simplicity, sensitivity, low detection limit, selectivity,speed, wide analytical range, instrumental availability, afford-ability, and possibility of on-site detection. These requirementsnarrow the acceptable range of analytical techniques to photo-metric and electrochemical methods. Photometric methods forthe detection of gold are based on the formation of color com-pounds that can be easily detected and quantified by absorptionspectroscopy or fluorometry, as in the classic example of forma-tion of the yellow-orange bromoaurate ion [8].

AuCl4− + 4Br− → AuBr4

− + 4Cl− (1)

With the obvious advantages of simplicity and speed of theanalysis, the major drawback of the photometric methods is thehigh minimum detection level (MDL).

The more sensitive electrochemical methods are based onanodic stripping voltammetry (ASV), which has served the sci-entific community well for the past 30 years for the detection ofultratrace amounts of metal ions in water samples. Among theadvantages of stripping voltammetry are a linear dynamic rangeoplctpapHo

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est results cannot be linked directly to pathogen removal (theAC particle size does not remain constant during the test, andhe particle size distribution may vary considerably, dependingn the manufacturer and the dispersion mode of the PAC and theater quality).The overall objective of the reported study was thus to

evelop an alternative detection technique for the quick, sim-le, and relatively inexpensive evaluation of the integrity ofembrane systems. With such a technique, the test membrane

ystem would be challenged with monodispersed tracer parti-les, and the concentration of tracer would be measured in theeed and in the permeate. The log removal value (LRV), defineds log10(Cin/Cout), would then be used as a direct indicator ofhe performance of the membrane system. Undetectable levels ofhe probe in the permeate would indicate that the membrane skinayer is not compromised to the degree that affects virus reten-ion. In the development of such a system, we chose two typesf tracer particles, citrate-stabilized (12 nm) or thiol-stabilized15 nm) gold nanoparticles or fluorescent-dye-labeled MS2 bac-eriophages (28 nm). The gold challenge test assumes that the

embrane retains the particles by a size-exclusion mechanismegardless of surface chemistry, vitality, pathogenesis, and otherharacteristics that distinguish organic from inorganic particles.old nanoparticles were preferred to other metal nanoparticlesue to the extremely low background concentration of gold inater systems (usually between 5 and 50 ppt, with an average

oncentration of about 13 ppt [6]). The idea of developing theabeled-phage probe was based on previous experiments show-ng that, in terms of behavior and permeability, the labeledhages resemble native phages [7], which are the best availableimics of potent pathogenic viruses. The labeling and count-

f four to five orders of magnitude, a low MDL in the singlearts per billion (and lower) range, precision of the response,ow cost of the instrumentation, absence of a matrix effect, andompactness. However, a drawback of applying ASV to ultra-race amounts of gold is the slow nucleation, which requiresrolonged run times. The usual solution to this problem is todd a precise amount of another metal with a considerably lowerositive oxidation potential, such as mercury [9], i.e., +0.4 V forg versus +0.8–0.95 V for Au [10]; thus, the voltammetric peaksf mercury and gold are easily resolved.

. Materials and methods

.1. General

Deionized (DI) water (electrical resistivity > 18 M� cm) wasbtained from a MilliQ water purification system. Whereequired, the pH of the phosphate buffer solution consistingf a 0.1 M solution of Na2HPO4 and KH2PO4 was adjustedith 0.01 M NaOH (Frutarom, Israel) or 0.01 M HCl (J.T. Bakerhemical Co., Philipsburg, NJ, USA). The exact pH values of

he buffer solutions were determined with an Accumet AB15H meter. All glassware used was treated with piranha solution3:7 hydrogen peroxide/concentrated sulfuric acid).

.2. The cell

A laboratory-scale 500-ml stirred cell (Spectrum Inc. Ltd.)as used (Fig. 1). The cell consisted of an acrylic glass cylinder

ble to withstand an internal pressure of ≤5 × 105 Pa (≤5 bar).he feed solution in the cylinder was stirred with a magnetictirrer, whose speed could be varied between 0 and 6.67 Hz

V. Gitis et al. / Journal of Membrane Science 276 (2006) 199–207 201

Fig. 1. Schematic representation of the filtration setup.

(400 rpm). The cell was fitted with a back-pressure controllerfor control of transmembrane pressure and provisions for 76-mm diameter membranes. Circular pieces of the membranesto be tested, approximately 150 nm thick, were supported on aTeflon base. The entire system was mounted on a stainless steelframe. The feed solution was pressurized by nitrogen at a con-stant pressure of 2.0265 × 105 Pa (2 atm) throughout the testingof the various membranes. Pressure was set by a pressure regu-lator at a desired set-point.

2.3. Membranes

Seven types of UF flat-sheet membranes of typical materialand size range for UF water treatment applications were usedin this study, i.e., cellulose acetate (CA-0.5, CA-5, and CA-10), cellulose ester (CE-20), polyvinylidene fluoride (PVDF-55), and polyethersulfone (PES-15 and PES-20) (Table 1). Allthe membranes were commercially available integrally skinnedasymmetric membranes that differ in terms of their chemical andmechanical stability and of their resistance to organic fouling.PVDF and PES membranes are usually considered more stablebut less resistant.

Contact angle – as an index of membrane hydrophobicity –and permeability of the pure membranes were measured prior

to the experiments. Contact angle was determined by the ses-sile drop method. In this test, a ∼20-�l drop of DI water wasplaced onto the dried membrane surface with a microsyringe,and the air/water-surface contact angle was measured with agoniometer (Rame-Hart, CA) within 10 s. Contact angle mea-surements were performed in triplicate using separate piecesof membrane. Contact angles greater than 90◦ indicate that themembrane is hydrophobic, and extra pressure should be appliedto push the water through the membrane. In the current study,the only hydrophobic membrane was PVDF-55. Polyether sul-fone membranes were, on average, more hydrophilic than thosemade of cellulose acetate. Membrane permeability to pure waterwas obtained by filtration of DI water through the membrane inthe test cell for 30 min at 1 × 105 Pa (1 bar) N2 pressure afterthe membranes had been soaked in DI water at 30 ◦C for 1 h toremove glycerin.

2.4. Experimental protocol

To determine the chemical stability (integrity) of the mem-brane samples, filtration experiments were performed withprobes of citrate- or thiol-stabilized gold nanoparticles (see Sec-tions 2.5–2.7) or of fluorescent-labeled MS2 bacteriophages (seeSection 2.8). The following filtration protocol was employedin all the experiments: 100 ml of feed solution consisting ofDI water spiked with 5.2 ppm of gold nanoparticles or 2 × 109

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Table 1P

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roperties of membranes used in the current study

ype Manufacturer MWCO (kDa) Average po

A-0.5 Spectruma 0.5 1.5A-5 Spectrum 5 4A-10 Spectrum 10 5E-20 Spectrum 20 9ES-15 FuMA-Techb 15 7ES-20 Sterlitechc 20 9VDF-55 FuMA-Tech 100 55

a Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA).b FuMA-Tech GmbH (St. Ingbert, Germany).c Sterlitech Corporation (Kent, WA, USA).

laque-forming units (PFU)/ml of labeled MS2 bacteriophagesere added to the stirred cell. Gold nanoparticles served as arime tracer due to their smaller probe size and higher (4.5) LRVensitivity, which was calculated in this case as Cf-max/MDL,here Cf-max is the maximum feed concentration (g/l) andDL = 0.2 �g/l, the minimum detection limit in the filtrate. The

ensitivity of dyed bacteriophages was somewhat lower, on therder of three LRV with relatively high MDL of 106 PFU/ml inistilled water (corresponding to signal-to-noise = 3). The exper-ments were performed at a constant temperature of 22 ± 1 ◦Cnd near neutral pH values of 6.7–6.8 (NaOH correction).

.5. Preparation of gold nanoparticles

A colloidal aqueous suspension of citrate-stabilized goldanoparticles was prepared by adding 4 ml of a 1% solution ofrisodium citrate to 40 ml of a 0.01% (w/v) solution of HAuCl4

e (nm) Contact angle, θ (◦)in water

Permeability (l/(×105 m2 h Pa)(l/m2 h bar))

95 ± 1 12 ± 292 ± 1 16 ± 390 ± 1 19 ± 388 ± 1 44 ± 551 ± 1 90 ± 252 ± 1 50 ± 3

106 ± 1 250 ± 7


[11]. The mixture was stirred for 5 min under gentle boiling,cooled to room temperature, and stored at 4 ◦C. The obtainedcolloids were composed of an internal core of gold metal coatedwith a layer of weakly bound acetone dicarboxylate and othercitrate oxidation products [12]. The colloids possessed a nega-tive surface charge in water as a result of partial ionization ofthe carboxylic groups, starting around pKa 3.1.

Another set of nanoparticles were stereo-stabilized with mer-captopropionate ions (thiol-stabilization) known for their abilityto form strong Au–S bonds [13,14]. To prepare these nanopar-ticles, the citrate-stabilized nanoparticle solution was stirredovernight with a solution of 0.01 mM 11-mercaptoundecanoicacid, resulting in the formation of a stable particle solution. Thesolution was dialyzed against DI water for 3 days to removecitrate and thiol debris.

2.6. Characterization of gold nanoparticles

The prepared nanoparticles were characterized in terms ofsize, zeta potential, and stability of the suspension. Size wasdetermined with a transmission electron microscope (TEM;Hitachi H-7000). The particle size distribution and zeta poten-tial were obtained with a ZetaPlus (Brookhaven InstrumentsCorporation, Holtsville, NY, USA) instrument equipped with a30 mW 657 nm laser (Hamamatsu Photonics K.K., HamamatsuCity, Japan). The stability of obtained colloidal solutions wasiiaM

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Fig. 2. Schematic representation of the electrochemical cell.

A Pt wire was used as the auxiliary electrode. The referenceelectrode [a saturated Ag/AgCl electrode (model 722, MetrohmAG, Herisau, Switzerland) immersed in dilute aqua regia] wasconnected to the cell through a salt bridge (Fig. 2). ASV wasperformed as follows. In the preconcentration and depositionstep, 5 ml of a water sample containing gold nanoparticles wasmixed with 5 ml of concentrated aqua regia. The gold was thenelectrodeposited by cathodic stripping at −0.7 V for 15 min. Toimprove the nucleation rate, the gold was co-deposited in thepresence of mercury. In the initial experiments, a sample ofgold solution containing 100 ppm Hg2+ was held at −1.0 V for30 s followed by 10 cycles each of −0.6 V for 10 s followed by+0.3 V for 1 s. The solution was stirred continuously at 15.83 Hz(950 rpm) with a magnetic stirrer. Stripping was performed bysix cycles of linear potential scan from −0.7 to 1.0 V at a scan-ning rate of 100 mV/s. All measurements were performed with acommercially available 263 A potentiostat–galvanostat (EG&GPrinceton Applied Research, NJ, USA). After the stripping step,the cell was rinsed twice with DI water and twice with diluteaqua regia (0.1 M HCl/0.4 M HNO3 in DI water), at least 10 sper wash. After rinsing, no residual peaks were detected withinthe potential range, and the electrodes could be used for subse-quent measurements. The charge required to strip the depositedgold was given by time integration of the current peak (Head-Start Software, Ruabon, England). Since in our tests close tofull recovery was attained, i.e., the quantity of stripped gold waseaawgS

2

pc

nspected over a period of 2 months on a weekly basis by track-ng changes in the UV–vis spectra of the colloidal solutions withCary E1 double-beam UV–vis spectrophotometer (Varian Inc.,ulgrave, Australia).To compare the citrate- and thiol-stabilized colloids, changes

n the zeta potential and size of the gold nanoparticles as a func-ion of pH were determined in 0.01 M phosphate buffer solutionsith HCl/NaOH pH correction in the range of 1–9.5.

.7. Quantification of gold nanoparticles

Gold nanoparticles were quantified spectrophotometricallynd electrochemically. The spectrophotometric analysis wasased on the determination of the bromoaurate ion. For thisnalysis, the sample was preconcentrated by centrifugation inn Optima Max Ultra Centrifuge (Beckman Coulter, Palo Alto,A, USA) at 333.33 Hz (20,000 rpm) for 3 min at 25 ◦C. Therecipitate was resuspended in 1 ml of DI water, with no visibleigns of change in the colloidal status. The visual observationsere confirmed by measurements of size distribution, which

howed a sharp peak at 13 nm, with no sign of aggregation. Theold colloid was dissolved in aqua regia (3 M HCl:1 M HNO3),ollowed by addition of NaBr to give the stable yellow-orangeomplex ion AuBr4

− (Eq. (1)).The ASV set-up for the electrochemical determination of

old nanoparticles in water samples consisted of six dispos-ble graphite electrodes (pencil leads, 7 cm long, 0.7 mm i.d.,acura Mines, Japan) connected together electrically with a cop-er strip to form an electrode assembly with a surface area of0 cm2 (Fig. 2). The assembly was suspended approximatelymm above the bottom of a 20-ml volume flat acrylic cell.

qual to the quantity of Au3+ ions in solution, the stripping peakrea was proportional to the amount of metal reduced in theccumulation step. The obtained stripping peak values agreedith those obtained with Faraday’s law to within an 8% mar-in. The ASV parameters were then optimized, as described inection 3.

.8. MS2 bacteriophages

Cultivation and counting of native MS2 bacteriophages wereerformed as previously described [7]. To prepare the fluores-ent bacteriophage probe, a titer of 2 × 1011 PFU/ml of MS2


bacteriophages was labeled with fluorescein-5-isothiocyanateby means of the procedure of Banks and Paquette [15] for theconjugation of amine-reactive probes to the l-lysine group ofmyoglobin. The labeled bacteriophage mixture was purified bymembrane dialysis (The Scientific Instrument Centre Ltd., Lon-don, UK) under stirring at 4 ◦C to remove low-molecular-weightorganic compounds and free dye molecules. Approximately,10 h of dialysis were needed to obtain a leak-free solution, butthe phages were dialyzed for at least 6 days to assure a leak-freesuspension. Comprehensive characterization [7] of the obtainedsuspension showed that the labeled probe maintains all the prop-erties of the native phages except for viability. Determination ofthe non-viable viruses was performed by fluorescence analysis.The fluorescence of labeled phages exhibited linear dependenceon the virus level before labeling [7].

3. Results and discussion

3.1. Characterization of gold nanoparticles

TEM micrographs gave the average diameter of the citrate-and thiol-stabilized gold particles as 12 ± 3 nm (Fig. 3a and b,respectively).

Fn

Fig. 4. (a) Zeta potential and (b) size distribution of gold nanoparticles as afunction of pH.

Measurements of the zeta potential of the citrate-stabilizedgold nanoparticles over a range of pH values indicated pos-itive charging of the nanoparticles in the pH range of 1–3(Fig. 4a). The charge on the gold particles shifted to nega-tive values in the pH range between 3 and 5, indicating par-tial ionization of carboxylic groups of the capping agents.A similar effect was observed for the thiol-stabilized parti-cles, for which ionization of the carboxylic functional groupsat pH values >5 contributed to negative zeta potential values(Fig. 4a). The negative surface charge on both thiol- and citrate-stabilized particles increased in the pH range of 5–7 and sta-bilized at zeta potential values of −30 mV as the pH increasedabove 7.

The degree of ionization of the citrate- and thiol-stabilizedparticles varied inversely with the average particle size (Fig. 4b).Although the average diameter of the thiol-stabilized parti-cles decreased with time, there was still some aggregation,since the average particle diameter remained in the area of80–120 nm. On the other hand, citrate-stabilized particles exhib-ited a uniform particle size distribution in the pH range of5–9.5.

Examination of the UV–vis spectrum of the citrate-stabilizedsolution that had been left to stand for 2 months showed it to berelatively stable, since only one peak (at 535 nm) was evident(Fig. 5): a shift in the peak or the appearance of a second peak

ig. 3. TEM micrographs of (a) citrate-stabilized and (b) thiol-stabilized goldanoparticles.

would have indicated decomposition or precipitation.

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The kinetics of decomposition of the colloidal gold uponntroduction of aqua regia was assessed with spectrophotome-ry. The results are depicted in Table 2. Here disappearance of35 nm peak indicated interconversion Au0 → Au3+, i.e. fromolloidal to ionic gold required for ASV application. The com-lete oxidation of Au particles was achieved with a ratheroncentrated aqua regia that contains 3 M of HCl and 1 MNO3.


Fig. 5. Absorption spectra of citrate-stabilized gold nanoparticles.

3.2. ASV

ASV was performed with a deposition potential of −0.6 V,a deposition time of 60 s, stirring rate of 6.67 Hz (400 rpm),a scan rate of 100 mV/s, a scan increase of 2 mV, an ini-tial stripping potential of −0.6 V, and vertex potential 1.3 V.With the exception of the scan increase (set at 1 mV), all theabove-mentioned parameters were optimized. Optimization ofthe deposition potential was performed for a solution contain-ing 10−7 g Au3+ for a constant accumulation time of 600 s.The deposition potential was varied between −0.4 and −1.0 V.Complete collection of gold from the solution was obtained atpotentials more negative than −0.7 V (Fig. 6a). To determinethe required deposition time, an experiment was run for 60 min,as shown in Fig. 6b. Since complete deposition appeared to beobtained at run times exceeding 10 min, a period of 15 min wasdeemed to be optimal for complete deposition of the gold fromthe sample for a working electrode area of 40 cm2. Other param-eters that required optimization were the size of stirrer bar (foundto be 2/3 by length of the diameter of the electrochemical cell)and the stirring rate (Table 3). The practical limit of the stir-ring rate was 15.83 Hz (950 rpm), since faster rates resulted insplashing.

Increasing the voltage scan rate during the stripping in therange of 0.5–100 mV/s resulted in a linear increase of the heightand a decrease of the width of the stripping peak.

bam

TD

T

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C

Fig. 6. (a) Effect of deposition potential on the stripping peak intensity; (b)effect of deposition time on the deposition of gold nanoparticles dissolved inaqua regia; (c) typical cyclic voltammogram of colloidal gold (10 ppm) in 1 Maqua regia.

distinguishable from the gold peak. Calibration plots for Au(III)are shown in Fig. 7. The graphs showed a linear relationshipbetween the charge (C) and the concentration of gold, with zerointercept and Pearson’s coefficients of R2 = 0.9997 and 1 for thelow and high concentration domains, respectively (Fig. 7a andb). No interference with mercury was detected.

The tests repeated with tap water (water quality for Jerusalem,Israel) gave identical slopes and intercepts for both the high

Table 3Dependence of gold accumulation on the stirring rate

Stirring rate (1/60 Hz (rpm)) Stripping peak (mC)

400 0.09600 0.11800 0.13950 0.145

1200 0.14

ASV conditions: Deposition time and potential = 15 min and −0.7 V vs. Ag/AgClsaturated, initial stripping potential −0.6 V, vertex potential +1.0 V, scan rate100 mV/s, scan increase 1 mV.

A single, sharp gold stripping peak was obtained at potentialsetween +0.8 and +1.0 V, depending on the concentration of goldnd the deposition interval (Fig. 6c). As can be seen in Fig. 6c,ercury was oxidized at +0.4 V, and the mercury peak was easily

able 2issolution of gold nanoparticles in aqua regia

ime (h) Aqua regia concentration (M)/color, ABS

0.5 M HCl 1 M HCl 2 M HCl 3 M HCl

mmediately 0.016 0.011 0.003 ND0.009 0.011 0.003 ND0.001 ND ND ND0.001 ND ND ND

onditions: 1 M HNO3; 50 ◦C. ND: below detection limit.


Fig. 7. (a) Calibration graphs for gold nanoparticles dissolved in aqua regia for(a) low 10−9 to 10−6 g and (b) high 10−6 to 10−4 g gold masses.

and the low concentration ranges. Similarly, no interference wasdetected with other metals.

3.3. Probe removal efficiency

The first set of filtration experiments was performed onuncompromised membranes to assess the membrane perfor-mance and the removal ratio. Fig. 8 shows the detected concen-trations of citrate- and thiol-stabilized gold nanoparticles in thepermeate as a function of pH. For the CA-10, CE-20, and PES-20 membranes, the concentration of gold in the permeate wasbelow the MDL of 0.2 �g/l. With a 5.2 mg/l initial concentration

Fvt(c

the log removal of gold was plotted as 4.5 LRV (curve c). For thePVDF-55 membrane, which has a much larger molecular-weightcutoff (MWCO) – and correspondingly a larger pore size cutoff– both citrate- and thiol-stabilized gold nanoparticles exhibitedsome degree of permeation. For the citrate-stabilized gold, theLRV ranged from 1.7 at pH 3 to 1.3 at pH values >7 (curve a). Theobserved increase in permeation of citrate-stabilized nanoparti-cles can be explained in terms of the changes in surface chargeand observed size cutoff of the different membranes: stronglyelectronegative and small (12 ± 2 nm average diameter) parti-cles increased permeate gold concentrations by 2.5% over a pHrange between pH 3 and 7. Thiol-stabilized gold exhibited a con-stant 3% penetration (1.5 LRV, curve b), which is understandablein view of the lack of acid–base functional groups on the surfaceof those particles.

The permeates were analyzed by means of three light scat-tering techniques: turbidity monitoring, particle counting, andparticle size distribution. Turbidity measurements (Ratio/RXturbidimeter Hach Company, Loveland, CO, USA) were foundto be not sufficiently sensitive for the study performed (dueto the red color of colloidal gold): initial turbidity readingswere on the order of 3.6 nephelometric turbidity units (NTU),and post-filtration values fell below the detectable level of 0.2NTU. The same trend was obtained with the 8011HIAC-RoycoLab Liquid Particle Counting System (Hach Ultra Analytics,Oregon, USA). Counts in the permeate fell below the detec-tPomt(tFad(

d

F0

ig. 8. Concentration of gold nanoparticles in the permeate after filtration witharious UF membranes at different pH values: (a) citrate-stabilized gold fil-ered through PVDF-55, (b) thiol-stabilized gold filtered through PVDF-55, andc) citrate-stabilized gold filtered through CA-10, CE-20, and PES-15. Initialoncentration = 5.2 mg gold/l, MDL = 0.2 �g/l.

ion limit of 30 particles/ml for all types of membrane, exceptVDF-55. For the latter membrane, a constant backgroundf 50–60 particles/ml was obtained. Particle size distributioneasurements on the feed solution gave a unimodal distribu-

ion of gold nanoparticles, with a sharp peak around 15 nmthe intensity-weighted distribution). The particles were unde-ectable in the permeate of all membranes except PVDF-55.or the permeate of PVDF-55, citrate-stabilized particles gavemultimodal size distribution with intensity-weighted effectiveiameter of 49.7 nm but also with a secondary peak at 656 nmFig. 9).

The experiment was also performed with both native andye-labeled MS2 bacteriophages (Fig. 10). The concentration of

ig. 9. Size distribution of gold nanoparticles in the permeate from the PVDF-55 membrane.


Fig. 10. Concentration of native and dye-labeled MS2 bacteriophages in thepermeate of various UF membranes as a function of pH values: (a) nativeMS2 bacteriophages filtered through PVDF-55, (b) fluorescent-labeled MS2bacteriophages filtered through PVDF-55 MS2, and (c) native/dyed MS2bacteriophages filtered through CA-10, CE-20, or PES-15. Initial concentra-tion = 2 × 109 PFU/ml, MDL = 106 PFU/ml.

the native MS2 bacteriophages in the permeate remained unde-tectable for all the membranes except PVDF-55. For the lattermembrane, application of 2.5 × 106 bacteriophages/ml resultedin the transition of 7.4 × 104 bacteriophages/ml (1.5 LRV, curvea) – as determined by the plaque-forming technique – to thepermeate. Similar results were obtained with dye-labeled bacte-riophages: the concentration of dye-labeled MS2 in the permeateranged between 0.2 and 0.4 fluorescence intensity units (FIU),with the initial concentration being 23.7–27.9 FIU, i.e., a tran-sition of the order of 1.5% (1.8 LRV, curve b), a value thatis somewhat lower than that for the native bacteriophages. Weattribute the differences in the results for the removal rates for thenative and dye-labeled bacteriophages to differences in the mea-surement techniques. The PFU technique regards aggregates ofbacteriophages – as may indeed exist in the feed solution – assingle plaque-forming units, whereas the fluorescence techniquegives identical weighting to all virus particles, whether they aremonodispersed or present in a cluster.

4. Discussion

The above-described experiments confirm that the majorityof conventional monitoring protocols are either unsuitable ornot sufficiently sensitive for UF membrane applications. Amongthe methods with limited sensitivity are light-scattering tech-nmtfipsaoq

tion or aggregation – as observed in Fig. 9 – may lead to falseinterpretation of the test results. Taking Fig. 9 as an example,without prior knowledge of the monosized distribution of thefeed one may have reached the erroneous conclusion that themembrane has >100 nm breaches.

The sensitivity of the both citrate- and thiol-stabilized goldspike probes, with ASV detection, was of the same order as thatfor the native MS2 bacteriophage spike with the plaque form-ing technique. Since the size of the gold nanoparticles differsfrom that of the bacteriophages, the two types of probes providecomplementary information. Although the native virus probewas suitably sensitive (nothing mimics viral transport as well asthe viruses themselves), spiking feed solutions with vital nativeviruses is problematic: possible complications include penetra-tion of vital viruses through the membrane rack; time (minimum24 h detection time) and labor demands; necessary safety pre-cautions for working with viral microorganisms; and possiblenoise from other viruses in the counting of non-selective hostbacteria (such as Escherichia coli). The fluorescent-dyed bac-teriophage probe was appreciably less sensitive than the othertwo probes. The fluorescent probe can therefore only be usedas a convenient tool for background analysis. To improve thesensitivity of the fluorescent probe, more sensitive techniquesare required for the detection of fluorescence.

Assuming that the main removal mechanism for goldnanoparticles is size exclusion, the gold-nanoparticle probeppohsd

dmfic(miwso1ttrsb(arrtits

iques, both monochromatic (i.e., laser-based) and multichro-atic (such as turbidity measurements). The drawbacks of these

ests are: their inability to distinguish between the removal ofeed particles and the removal of parasites, their inability to mon-tor the passage of viruses, and the high minimum detectablearticle size (especially for particle counting). The relativelyensitive particle size distribution technique is similarly not suit-ble, since it merely indicates the presence or absence of particlesf certain size ranges in a water sample, but it cannot provideuantitative data. Moreover, post membrane particle disintegra-

romises the following advantages: low detection limit, sim-le and sensitive on-site detection, very low background levelf the probe moiety in natural systems, the ability to produceighly concentrated stock solutions of the probe to facilitate on-ite spiking, non-pathogenicity and safe use in drinking wateristribution systems, high monodispersity, and affordable price.

The desirable limit of detection of the gold nanoparticleseserves some additional discussion in connection with the ulti-ate target—detection of breaches in a full size membraneltration system. For that we can discuss the following typi-al filtration plant of unspecified size. Since membrane systemsparticularly RO membranes) are constructed in autonomousembrane modules the actual capacity of the target plant is

mmaterial, and we may consider a test of a filtration modulehich is comprised of some 160 cells/module. Each module

hould be tested separately. A typical module should have anperational capacity of some 20,000 m3/day corresponding to0,000 m3/day of treated water. For the sake of these calcula-ions, the peak width at half height (PWHH) of the responseo the introduction of a slug of tracers (nanoparticle probe) isoughly estimated to be of the order of 0.05 h. This involvesome overestimate that takes into account mixing in the pipesefore and after the membrane unit. Please note the following:1) the residence time in a module itself is only a fraction of

minute. (2) This calculation does not imply that the meanesidence time in the filtration unit is 3 min. In fact the meanesidence time will be much larger than the PWHH. Even forhe unreasonable case of ideal stirred tank configuration, PWHHs roughly 70% of the mean residence time. Suppose further thathe test is conducted by introduction of 1 l of an aqueous disper-ion of gold nanoparticles at a weight fraction W = 0.0001 (i.e.,


0.01 wt%). This implies that the detection limit of the gold at theoutlet should be lower than the method detection limit (MDL):

MDL =(

W × 1000

Mw

)×

(1

PWHH × (F/24)

)

× (10−LRV) = 1.2 × 10−9 M

where W is the gold fraction by weight = 0.0001 (dimension-less), Mw the molecular weight, PWHH the peak width athalf height = 0.05 h, F the feed flow rate entering a mod-ule = 20,000 m3 day, and LRW is the target log reduction = 4.

Thus, an MDL of 1 nM of gold in the outlet should be suf-ficient for detection of breaches, provided that the backgroundlevel of gold is sufficiently low. Fortunately, the gold concentra-tion in the Israeli taps (and elsewhere) is way lower than that,and our demonstrated detection limit is an order of magnitudelower. For comparison between the two types of probes that wereexamined in this study please note that a concentration of 0.1 of10 nm gold probes corresponds to 250,000 particles/ml. Quot-ing the Nobel Laurate Richard Feynman “there is a lot of roomat the bottom” and by appropriate optimization of the detectionlimit one may anticipate at least one or two orders of magnitudeincrease of the LRV and a corresponding reduction of the goldprobe consumption.

5. Conclusions

aouvd

A

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for providing a purified solution of MS2 bacteriophages. Theviews expressed in this paper are those of the authors and do notnecessarily reflect the views of the U.S. EPA.

References

[1] EPA, Membrane Filtration Guidance Manual, U.S. Environmental Pro-tection Agency Office of Water (4601), EPA 815-d-03-008, June 2003.

[2] M.E. Walsh, M.P. Chaulk, G.A. Gagnon, Indirect integrity testing on apilot-scale UF membrane, J. Water Supply Res. Technol.—AQUA 54(2005) 105–114.

[3] K. Farahbakhsh, Monitoring the integrity of low-pressure membranes,J. Am. Water Works Assoc. 95 (2003) 95–107.

[4] L.B. Landsness, Accepting MF/UF technology—making the final cut, in:AWWA Membrane Technology Conference Proceedings, San Antonio,TX, March, 2001.

[5] S.C.J.M. van Hoof, L. Broens, A. Narstedt, S. Panglish, R. Gimbel,Development of a new integrity testing system, in: Proceedings of theIWA/AWWA Conference Membranes in Drinking and Industrial WaterProduction, Mulheim an der Ruhr, Germany, September, 2002.

[6] B. Remy, I. Verhaeghe, P. Mauchien, Real sample analysis by ETA-leafswith background correction—application to gold determination in riverwater, Appl. Spectrosc. 44 (1990) 1633–1638.

[7] V. Gitis, A. Adin, A. Nasser, J. Gun, O. Lev, Fluorescent dye labeledbacteriophages: a new tracer for the investigation of viral transport inporous media. 1. Introduction and characterization, Water Res. 36 (2002)4227–4234.

[8] W.A.E. McBryde, J.H. Yoe, Colorimetric determination of gold as bro-moaurate, Anal. Chem. 20 (1948) 1094–1099.

[9] H. Huiliang, D. Jagner, L. Renman, Computerized flow constant-current

[

[

[

[

[

[

This paper presents a new method that may facilitate fastnd accurate evaluation of membrane integrity, both in the lab-ratory and in the field. The proposed methodology provides anique opportunity for the development of an on-line test forirus removal that can comply with the stringent regulations forrinking water treatment.

cknowledgements

This study was supported by the U.S. Environmental Protec-ion Agency, National Risk Management Research Laboratory,

ater Supply and Water Resources Division. The authors thankr. Boris Anisimov, of The National Physical Laboratory, Israel,

or his help in comparative gold detection analysis. The authorsre also grateful to Dr. Nasser, from the Israel Ministry of Health

stripping analysis for gold(III) with carbon or platinum fibre electrodes,Anal. Chim. Acta 208 (1988) 301–306.

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13] M. Brust, J. Fink, D. Bethell, D.J. Schiffrin, C. Kiely, Synthesis andreactions of functionalized gold nanoparticles, J. Chem. Soc. Chem.Commun. 16 (1995) 1655–1656.

14] K. Aslan, V.H. Perez-Luna, Surface modification of colloidal gold bychemisorption of alkanethiols in the presence of a nonionic surfactant,Langmuir 18 (2002) 6059–6065.

15] P.R. Banks, D.M. Paquette, Comparison of 3 common amine reactivefluorescent-probes used for conjugation to biomolecules by capillaryzone electrophoresis, Bioconjugate Chem. 6 (1995) 447–458.

Nanoscale probes for the evaluation of the integrity of ultrafiltration membranes

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