Biological Contamination of Nanoparticles and Its Manifestation in

Letter pubs.acs.org/ac

Biological Contamination of Nanoparticles and Its Manifestation in Optical Absorbance Measurements Yuriy Garbovskiy* UCCS BioFrontiers Center and Department of Physics, University of Colorado Colorado Springs, Colorado Springs, Colorado 80918, United States ABSTRACT: The biological contamination of nanomaterials is a serious problem hampering their widespread use in biomedical products. Existing commercial chromogenic assays for the detection and quantification of biocontaminants such as endotoxin can interfere with nanoparticles thus leading to unreliable data. The results reported in this Letter offer a solution to the aforementioned problems of the nanoparticle interference and correct quantification of biocontaminants by decomposing their optical absorbance into two physically measurable components and analyzing them as a function of the concentration of contaminated nanoparticles.

T

detection even more.5 In both cases, standard chromogenic assays can yield either a false positive test (endotoxin-free nanoparticles are detected as contaminated with endotoxin) or incorrect values of the endotoxin level.12−15 As a result, in a general case, existing chromogenic bioassays are not universally applicable to detect and quantify the biocontamination of nanoparticles.5,8,9,13,15 Given the growing impact of nanotechnology on health care, pharmacy, and medicine, the need for the development of reliable methods applicable to detect the biological contamination of nanoparticles is very urgent.14,15 This Letter provides a general analysis of the optical absorbance of contaminated nanoparticles dispersed in the dispersion medium. Nanoparticles are assumed contaminated with biocontaminants prior to dispersing them in contaminant-free dispersion medium. As will be shown later on in this Letter, the quantification of the biological contamination of nanoparticles, free off any interference, can be achieved by decomposing their optical absorbance into two measurable components and analyzing them as a function of the concentration of nanoparticles. Consider a colorimetric method for the determination of the biological contaminant using a bacterial lipopolysaccharide (endotoxin) as a typical example of such contamination.16−18 Assuming the validity of the Beer−Lambert law, the optical absorbance (or optical density) can be written as eq 1:

he biological contamination of nanoparticles can easily happen at any stage of their production and handling. The most commonly observed biocontaminants are represented by endotoxins.1−3 Endotoxins (or bacterial lipopolysaccharides) are large (their molecular weight is 200−1000 kDa) and heatstable molecules which are the major component of the Gramnegative bacteria cell wall.4 An exposure to endotoxins can lead to a variety of biological effects including fever, septic shock, and even death.4,5 Existing methods to assess the endotoxin contamination include the rabbit pyrogen test, the Limulus Amoebocyte Lysate (LAL) test, and various types of biosensors.5−7 The chromogenic LAL test is among the most sensitive optical techniques to detect and quantify endotoxins. This test is based on the enzymatic reaction induced by endotoxin. The final product absorbs light in the visible spectrum (typically, measurements are taken at 405 nm) and the measured optical absorbance is proportional to the concentration of endotoxin. As a result, the quantification of endotoxin can be done by comparing the measured values of the optical absorbance to a standard curve of a “known” endotoxin amount.6,7 Engineered nanomaterials such as metal nanoparticles (silver and gold1,8) silica nanoparticles,9 metal oxide nanoparticles (Fe3O4,8 TiO210), and carbon-based nano-objects (graphene and carbon nanotubes11) are typically contaminated with endotoxin.5 The detection of endotoxin in the presence of nanoparticles is much more challenging.5,12 Even optically transparent nanomaterials can interfere with standard chromogenic bioassays developed to detect endotoxin.5,8,9,13 As was reported by many independent research groups, this interference can be caused by the activation of the chromogenic bioassay through the adsorption of some of its components onto the surface of nanoparticles.5,8,9,12,13 Moreover, the absorbance of light by nanoparticles complicates the endotoxin © XXXX American Chemical Society

α ODBIO = σBIO nBIOd log e

(1)

where σαBIO is the extinction cross section of the biospecies, nBIO is their volume concentration, and d is the optical path length. By comparing the measured value of the optical absorbance to Received: May 10, 2017 Accepted: June 26, 2017 Published: June 26, 2017 A

DOI: 10.1021/acs.analchem.7b01766 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry

nanoparticles. However, as was already mentioned above, even optically transparent nanomaterials can interfere with standard chromogenic assays developed to detect biocontaminants such as endotoxin.5,8,9,15 A possible way to overcome this problem can be found by considering the extinction coefficient of biocontaminants as a free ads sum of two components, αBIO = αads BIO + αBIO, where αBIO is the extinction coefficient of biocontaminants adsorbed onto the surface of nanoparticles, and αfree BIO is the extinction coefficient of biocontaminants freely dispersed in the transparent dispersion medium (in other words,αads BIO refers to the biocontaminant bound to the surface of nanoparticles, and αfree BIO describes nonbound biocontaminants). This consideration always holds true for the following reason. If contaminated nanoparticles are dispersed in a fluid carrier (dispersion medium), the redistribution of biocontaminants between the dispersion medium and nanoparticles takes place. The conservation law of the total number of biocontaminants can be written as eq 2:

the standard (calibration) curve, the concentration of the biological contaminants can be easily determined.16−18 The product, σαBIOnBIO, determines the optical extinction coefficient, αBIO, of the biological contaminant. Consider spherical nanoparticles contaminated with biological species-contaminants. This contamination can be quantified by means of the dimensionless contamination factor, νBIO. The contamination factor equals a ratio of the adsorption sites occupied by contaminants to the total number of all adsorption sites on the nanoparticle’s surface.19 If σNP S is the total surface density of all adsorption sites of a single nanoparticle, the concentration of biocontaminants carried by nanoparticles can be written as nBIO = nNPANPσNP S νBIO, where nNP is the volume concentration of nanoparticles;ANP is the surface area of a single nanoparticle; and νBIO is the aforementioned contamination factor. In this case the extinction coefficient of biocontaminant is proportional to both the concentration of nanoparticles and the contamination factor, αBIO = σαBIOnNPσNP S ANPνBIO. This dependence shown in Figure 1 can be used for the quantitative evaluation of the biocontamination of nanoparticles (The physical parameters used in the simulations are listed in Table 1).

0 free nBIO + nNPANPσSNPνBIO = nNPANPσSNPΘBIO + nBIO

where is the volume concentration of biological contaminants in a fluid carrier prior to mixing it with contaminated nanoparticles (n0BIO = 0 m−3 is assumed); the free meaning of nNP, ANP, σNP S , and νBIO is explained above; nBIO is the volume concentration of “free” (not bound to the surface of nanoparticles) biological species; ΘBIO is the fractional surface coverage of contaminated nanoparticles dispersed in a fluid carrier. At equilibrium, the fractional surface coverage ΘBIO can be written in the form of the Langmuir adsorption isotherm expressed by eq 3: ΘBIO =

Figure 1. Dependence of the extinction coefficient of biological contaminants on the weight concentration of nanoparticles. Physical parameters used in simulations are listed in Table 1. The values of these parameters were selected to reasonably represent existing materials.16−22

physical quantity

radius of nanoparticles, RNP, nm mass density of nanoparticles, ρNP kg/m3 mass density of the dispersion medium, ρ kg/m3 constant KNP−BIO, m3 total surface density of the adsorption −2 sites, σNP S , m contamination factor, νBIO

value 2 × 10−18 (Figures 1−4) 0 (Figures 1and 2) 2 × 10−19 (Figure 3) 10−15 (Figure 4) 10 (Figures 1−4) 10490 (Figures 1−4) 1000 (Figures 1−4) 10−21 (Figures1−3,4a) 1016 (Figures 1−4) 10−4; 10−3; 10−2 (Figure 1) 10−2 (Figures 1−4)

In calculations shown in Figure 1 the weight concentration of ρ 1 nanoparticles, ωNP, is used (nNP ≈ ω NP ρ V , where VNP is the NP

free KNP−BIOnBIO free 1 + KNP−BIOnBIO

(3)

where KNP−BIO is the constant describing the adsorption− desorption process of biocontaminants onto/from the surface of nanoparticles.19 By solving eqs 2 and 3 for nfree BIO, the absorption coefficients of both adsorbed and “free” biocontaminants can be calculated as α NP free α free αads BIO = σBIOnNPANPσS ΘBIO and αBIO = σBIOnBIO, respectively. ads free These extinction coefficients, αBIO and αBIO, along with their free sum, αBIO = αads BIO + αBIO, are shown in Figure 2 as a function of the weight concentration of nanoparticles, ωNP. free Interestingly, both αads BIO and αBIO exhibit nonlinear dependence on the concentration of nanoparticles, ωNP (Figure 2a, dotted and dashed curves). However, their sum αBIO = αads BIO + αfree is a linear function of ω (Figure 2a, solid curve). BIO NP According to Figure 2a, the use of αfree BIO for the quantification of the biocontamination of nanoparticles is especially advantageous at low concentrations, ωNP. An increase in the concentration of nanoparticles results in the saturation of the α extinction coefficient, αfree BIO → σBIOνBIO/KNP−BIO (1 − νBIO) ≈ α σBIOνBIO/KNP−BIO (νBIO ≪1). In the regime of low concentrations of nanoparticles αfree BIO can be approximated as a linear function of the concentration of nanoparticles, αfree BIO ∼ ωNP (Figure 2b, dashed curve) whereas ads 2 αads BIO can be expressed by a quadratic dependence, αBIO ∼ ωNP (Figure 2b, dotted curve). The major result shown in Figure 2 is the possibility to use free αBIO for the quantification of the biocontamination of nanomaterials. Experimentally it can be achieved by dispersing contaminated nanoparticles in the dispersion medium, applying

Table 1. Physical Parameters Used in the Modeling extinction cross-section, σαBIO, m2 extinction cross-section, σαNP, m2

(2)

0 nBIO

NP

volume of a single nanoparticle, ρ and ρNP are the mass density of the dispersion medium and nanoparticles, respectively). A linear dependence αBIO(ωNP) depicted in Figure 1 was calculated neglecting the absorption/scattering of light by B

DOI: 10.1021/acs.analchem.7b01766 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 3. (a) Dependence of extinction coefficients of biological contaminants, αBIO, and nanoparticles, αNP, on the weight concentration of nanoparticles, ωNP. (b) Dependence of the extinction coefficient of biological contaminants bound to the surface of nanoparticles, αads BIO, the extinction coefficients of nanoparticles, αNP, and their sum, αads = αNP + αads BIO, on the weight concentration of nanoparticles, ωNP. Physical parameters used in simulations are listed in Table 1.

Figure 2. Dependence of the extinction coefficient of biological contaminants bound to the surface of nanoparticles, αads BIO, freely ads free dispersed in the dispersion medium, αfree BIO, and their sum, αBIO + αBIO, on the weight concentration of nanoparticles, ωNP. Physical parameters used in simulations are listed in Table 1.

the centrifugation, and taking optical absorbance measurements of the supernatant collected at different concentrations of nanoparticles. In this case any available chromogenic bioassay can be used to assess the concentration of biocontaminants in the collected supernatant. An actual level of the biocontamination of nanoparticles (νBIO, σNP S ANPνBIO) can be obtained through the analysis of the measured experimental data points, αfree BIO(ωNP), using eqs 1−3. If nanoparticles do not interfere with chromogenic bioassays, free the use of the total extinction coefficient, αBIO = αads BIO + αBIO, is more convenient option. However, the absorption of light by nanoparticles can interfere with the used bioassay. In this case, the measured extinction coefficient α is a sum of the extinction coefficient of biocontaminants, αBIO, and the extinction coefficient of nanoparticles, αNP. The extinction coefficient of nanoparticles can be calculated as αNP = σαNPnNP, where σαNP is the extinction cross section of nanoparticles. The combined effect of nanoparticles and biocontaminants on the optical absorbance measurements is shown in Figure 3. To compare the case of nonabsorbing (Figure 2) and light absorbing (Figure 3) nanoparticles, the same contamination factor is used, νBIO = 10−2. Absorption of light by nanoparticles increases the measured extinction coefficient, α = αNP + αBIO. As a result, for the correct quantification of the biocontamination of nanoparticles, their contribution to the measured value of α should be taken into account (Figure 3a). If nanoparticles interfere with a chromogenic assay used to assess their biocontamination, the extinction coefficient of biocontaminants, αBIO, should be ads free decomposed into two components, αBIO and αBIO . The dependence αfree (ω ) is the same as that shown in Figure 2 BIO NP (dashed curve) and it can be used for the quantification of biocontamination of nanoparticles. The absorption of light by the adsorbed biocontaminants, αads BIO (Figure 3b, dashed curve), should be considered with the absorption of light by

nanoparticles, αNP (Figure 3b, dotted curve), since experimentally measured quantity in this case is the combined extinction coefficient expressed by αads BIO + αNP (Figure 3b, solid curve). Interestingly, at relatively low concentrations of nanoparticles, ωNP, the measured quantity, αads BIO + αNP, is a nonlinear function of ωNP. Figure 3 refers to the case of relatively low extinction coefficients of light absorbing nanoparticles, thus the following inequality holds true: αNP < αBIO. However, an opposite scenario, αNP ≫ αBIO, is also commonly observed. This case is shown in Figure 4. According to Figure 4a, relatively strong absorption of light by nanoparticles (solid curve) as compared to the light loss due to biocontaminants (dashed curve) makes an approach to quantify the biocontamination of nanoparticles by means of expression α = αBIO + αNP impractical. The only way to assess the biocontamination of nanoparticles is to decompose the extinction coefficient of biocontaminants into two terms, αBIO = free free αads BIO + αBIO, and use the second term, αBIO, for the analysis. As was already mentioned, this extinction coefficient, αfree BIO, is easily measurable physical quantity. Once measured as a function of the concentration of nanoparticles, it can be related to the actual values of their biocontamination factor by applying eqs 1−3. From perspectives of an experimentalist, it is important to note a strong dependence of the measured value, αfree BIO, on the constant KNP−BIO (Figure 4b). Figure 4b indicates that by changing this constant it is possible to increase the measured extinction coefficient (its saturation value is given by free αBIO →

α σBIO νBIO ). KNP−BIO

The constant KNP−BIO strongly depends on

the temperature and type of materials used in experiments.20 Therefore, at a given type of nanoparticles, it can be controlled C

DOI: 10.1021/acs.analchem.7b01766 Anal. Chem. XXXX, XXX, XXX−XXX

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Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. ORCID

Yuriy Garbovskiy: 0000-0003-3047-8761 Notes

The author declares no competing financial interest.

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ACKNOWLEDGMENTS The author would like to acknowledge the support provided by the UCCS BioFrontiers Center at the University of Colorado Colorado Springs.

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Figure 4. (a) Dependence of extinction coefficients of biological contaminants (both bound to the nanoparticles and freely dispersed in the medium), α BIO , and nanoparticles, α NP on the weight concentration of nanoparticles, ωNP. (b) Dependence of the extinction coefficient of biological contaminants freely dispersed in the dispersion medium, αfree BIO, on the weight concentration of nanoparticles, ωNP. Physical parameters used in simulations are listed in Table 1.

by varying the type of solvent (dispersion medium) and/or temperature. Some limitations of the proposed approach should also be mentioned. The discussion presented in this Letter assumes (i) a steady-state condition expressed by eq 3 and (ii) no aggregation of nanoparticles. However, it can also be extended to the nonstationary (transient) case by replacing eq 3 with a more general rate eq 4: free dnBIO a free NP = −kNP −BIOnBIOnNPANPσS (1 − ΘBIO) dt d NP + kNP −BIOnNPANPσS ΘBIO

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(4)

where kaNP−BIO is the adsorption rate constant, and kdNP−BIO is the desorption rate constant. These two constants define the constant KNP−BIO of eq 3 as KNP−BIO = kaNP−BIO/kdNP−BIO. The first term of the eq 4 describes the adsorption of biocontaminants on the surface of nanoparticles, and the second term accounts for their desorption from the surface of nanoparticles. It can be seen that eq 3 is a steady-state version dn free

= 0). of the eq 4 ( dBIO t The results presented in this Letter indicate that the biocontamination of nanoparticles can be assessed by analyzing the optical absorbance of contaminated nanoparticles dispersed in the dispersion medium as a function of their concentration. The key elements of this approach are (i) the evaluation of the extinction coefficient of biocontaminants which are not bound to the surface of nanoparticles (this eliminates the problem of nanoparticle interference), and (ii) the use of eqs 1−3 to assess the level of the biocontamination of nanoparticles. D

DOI: 10.1021/acs.analchem.7b01766 Anal. Chem. XXXX, XXX, XXX−XXX