Biosensors and Bioelectronics 55 (2014) 242–248
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Facile colorimetric method for simple and rapid detection of endotoxin based on counterion-mediated gold nanorods aggregation Yashan Wang 1, Daohong Zhang 1, Wei Liu, Xiao Zhang, Shaoxuan Yu, Tao Liu, Wentao Zhang, Wenxin Zhu, Jianlong Wang n College of Food Science and Engineering, Northwest A&F University, Yangling 712100, Shaanxi, China
art ic l e i nf o
a b s t r a c t
Article history: Received 13 September 2013 Received in revised form 18 November 2013 Accepted 2 December 2013 Available online 11 December 2013
Existence of endotoxin in food and injection products indicates bacterial contaminations and therefore poses threat to human health. Herein, a simple and rapid colorimetric method for the effective detection of endotoxin in food and injections based on counterion-mediated gold nanorods aggregation is first proposed. By taking advantage of the color change of unmodified gold nanorods resulted from endotoxin mediated gold nanorods aggregation, endotoxin could be detected in the concentration range of 0.01–0.6 μM. Further, we studied the performance of gold nanorods with different aspect ratios (2.7 and 3.3) in determination of endotoxin and found that gold nanorods with higher aspect ratio (AR) showed superiority in the sensing sensitivity of endotoxin. A good specificity for endotoxin, a detection limit of 0.0084 μM and recoveries ranging from 84% to 109% in spiked food and injection samples are obtained with the colorimetric method. Results demonstrate that the present method provides a novel and effective approach for on-site screening of endotoxin in common products, which is beneficial for monitoring and reducing the risk of bacterial contaminations in food and injections production. & 2013 Elsevier B.V. All rights reserved.
Keywords: Endotoxin Gold nanorods Colorimetric detection
1. Introduction Endotoxin, covering the adhesion peptide of the Gram-negative bacteria outer membrane, is a significant contaminant in both food industry and pharmaceutical production (Kim et al., 2006). The prototypical example of endotoxin in most bacteria is lipopolysaccharide (LPS), which is in fact the hazard of endotoxin. LPS consists of a large polysaccharide chain of repeating oligosaccharide subunits, a core oligosaccharide and a unique structure of lipid moiety, known as lipid A, which is mainly responsible for the biological toxicity, causing various effects on human, including fever, diarrhea, vomiting, septic shock, inflammatory reaction, low blood pressure and disseminated intravascular coagulation (Kim et al., 2006; Limbut et al., 2007). Septic shock induced by endotoxin is a significant threat to public health with more than 150 thousand related infection cases reported every year in the United States alone. Worldwide, the situation is not optimistic, especially in areas without sufficient sterilization and water treatment facilities (Uzarski and Mello, 2012). Large amounts of endotoxin can be mobilized only when the bacteria dissolved due to aqtocytolysis or artificially damaged (Cho et al., 2012), as a
n
Corresponding author. Tel.: þ 86 29 8709 2275; fax: þ 86 29 8709 2275. E-mail address: [email protected] (J. Wang). 1 Both of Yashan Wang and Daohong Zhang rank the first authors.
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.006
result, massive existence of endotoxin in water or drugs implies severe bacterial contamination. Although endotoxin is released after death of bacterial cells, it can survive continuously heating for 1 h at 100 1C. Therefore, it is intractable to control and remove in both food and drug manufacture procedures and environmental disinfection. On account of this, it is necessary to detect and remove endotoxin in food and drug production process as well as the final merchandise, as even small amounts of endotoxin will pose a serious threat to human health. Thus, various approaches are put forward for this purpose. Endotoxin is commonly quantified via the Limulus Amebocyte Lysate (LAL) assay based on an enzymatic coagulation of the blood of a primitive marine arthropod, the horseshoe crab, in the presence of endotoxin. This method, while sensitive, suffers from environmental sensitivity (pyrogen in water, pH and temperature fluctuation) as well as false positive responses to other carbohydrate-like molecules, such as β-glucan (Uzarski and Mello, 2012; US FDA, 1987). Besides, it has limited ability to detect inactive endotoxin in the system (Cheng et al., 2012). More importantly, LAL is extracted from the horseshoe crab, which is on the brink of extinction, and authorities have promulgated laws to protect them from overwhelming fishing (Sun et al., 2012). To break the ice, several new analytical techniques have been reported. A colorimetric endotoxin sensor prepared by functionalized polydiacetylene liposomes to provide differential responses to specific LPS strains was reported (Rangin and Basu, 2004). Then,
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fluorescently labeled analogs of two peptide variants derived from the LPS-binding protein CD14 were developed to detect and discriminate endotoxin (Voss et al., 2007). In addition, antimicrobial peptides, together with surface plasmon resonance were employed to detect and classify different lipopolysaccharide molecules (Uzarski and Mello, 2012). Besides, the improved SDS-PAGE technique is also available to detect LPS sensitively (Redwan, 2012). An innovatively designed method to detect endotoxin taking advantage of cysteamine-modified gold nanoparticles was proposed. It was the first attempt to determine endotoxin by nanostructure of noble metal which received a detection limit as low as 0.33 nM and applied to recognize the surface of Escherichia coli (Sun et al., 2012). Except for this, an electrochemical detection of LPS using a combination of ferrocene-attached polymyxin B and a modified glassy carbon electrode (Iijima et al., 2011) was carried out, after which, amine-terminated aptamer immobilized gold electrode was formed as an electrochemical endotoxin specific biosensor (Su et al., 2012). However, these methods are either expensive, labor intensive or time consuming, making them unsuitable for on-site analysis. Therefore, it is imperative to develop an easy-handled, cost-effective and environmentallyfriendly approach for endotoxin on-spot detection. In the past decades, gold nanorods (AuNRs) have been employed for a range of applications, including biosensing (Yu and Irudayaraj, 2007; He et al., 2008), cell imaging (Wang et al., 2009), medical diagnostics (Truong et al., 2011), and cancer photothermal therapy (Wang et al., 2011). AuNRs have been proved to be versatile and tunable materials compared to other materials including spherical nanoparticles, especially in the detection field due to the shape anisotropy of nanorods. They possess two directional electron oscillations in response to the polarization of the incident light, the transverse plasmon band and the longitudinal plasmon band, corresponding to light absorption and scattering along the short axis of the nanorods, and the long axis respectively. Particularly, the longitudinal absorption band is extremely sensitive to the changes in the dielectric properties of the surroundings, such as solvents, adsorbates, and the interparticle distance of the gold nanorods owing to localized surface plasmon resonance (LSPR) (Ho et al., 2012; Chen et al., 2013). Nevertheless, an absorption coefficient of the peak at the longitudinal plasmon band is quite large and this absorption decreases remarkably upon aggregation (Kitazaki et al., 2012), and even, in some cases, a new red-shifted absorption band would occur gradually. At the same time, a change of the color of AuNRs suspension can be observed due to the shift of absorption band. These properties suggest gold nanorods could enable highly sensitive, specific and visualized detection of biomolecules based on the aggregation mechanism (Niidome et al., 2010). For this reason, gold nanorods, modified or unmodified, as a powerful tool have been utilized to detect and monitor diverse targets, including amino acids (Sudeep et al., 2005; Wang et al., 2012), proteins (Zhu et al., 2011; Xu et al., 2012; Yasun et al., 2012; Zhang and Shen, 2013), multiple pathogens (Wang and Irudayaraj, 2008), different metal ions (Huang et al., 2013; Wang et al., 2013; Liang et al., 2012; Anand et al., 2013), disease markers (Truong et al., 2011; Huang et al., 2012) and some other chemical substances. However, to the best of our knowledge, strategy based on the aggregation of gold nanorods has not been reported for the selective sensing of endotoxin. Herein, for the sake of reducing microbial contaminants and toxins in food, decreasing harm caused by bacteria to humanity, we report a method for rapid and visualized detection of endotoxin in food and injections taking advantage of the counterion electrostatic interaction between endotoxin and unmodified gold nanorods. Furthermore, we explored the performance of gold nanorods with different aspect ratios (the ratio between the length and diameter of the rods) in sensitivity of quantitative
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determination. And the selectivity of this assay was also investigated. Finally, to validate the feasibility of the proposed method, several beverages, including milk, carbonic acid drink, and glucose injection were chosen to test the accuracy of measurements. We thus performed a rapid and reliable assay for the detection of the endotoxin level in food and drugs, and providing a possibility for improving the current situation of food and drugs contamination by bacteria.
2. Experiment 2.1. Chemicals and materials Chloroauric acid (HAuCl4 3H2O) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Silver nitrate (AgNO3), Cetyltrimethylammonium bromide (CTAB), and Lipopolysaccharide (LPS), which is extracted from E. Coli 055:B5, was purchased from Sigma (St. Louis, USA). The LPS molecular weight was measured to be 10 kDa in previous work (Sun et al., 2012). All reagents were at least of analytical reagent grade and used as received. Ultrapure water (18.2 MΩ cm), obtained from a water purification system, was used in the whole experiment. All the glassware was cleaned with aqua regia and thoroughly rinsed with ultrapure water before use. 2.2. Instrumentation The absorption spectra were recorded on a UV-2550 spectrophotometer (Shimadzu, Japan) with 1 cm pathlength cells at room temperature. Transmission electron microscopy (TEM) measurements were performed on a HT7700 (Hitachi, Japan) at 80 kV. 2.3. Synthesis of gold nanorods AuNRs were fabricated following the silver ion-assisted seedmediated and CTAB surfactant-directed method according to previous report (Nikoobakht and El-Sayed, 2003; Fu et al., 2012) with necessary modifications. Typically, the seed solution was prepared by the addition of HAuCl4 (0.6 mM, 2.5 mL) into CTAB (0.2 M, 2.5 mL) with gentle mixing. Subsequently, to the mixture solution, freshly prepared ice-cold NaBH4 solution (0.01 M, 0.3 mL) was added quickly, and the color of the solution changed from dark yellow to brown under vigorous stirring for 2 min. The obtained solution was kept as the seed solution and used within 4 h. Gold nanorods were prepared using an aqueous growth solution of CTAB (0.1 M, 40 mL) in a 50 mL plastic tube. To this solution, HAuCl4 (50 mM, 0.6 mL) and AgNO3 (50 mM, 0.03– 0.15 mL) were added with gentle inversion. The addition of different volumes of AgNO3 produces gold nanorods with various aspect ratios (Nikoobakht and El-Sayed, 2003). Then 0.5 mL of 0.08 M ascorbic acid was added, followed by a color change of the solution from dark yellow to colorless. Finally, 0.8 mL of seed solution was added to the growth solution and gently shaken for 10 s. The solution was left undisturbed overnight at 28 1C to ensure fully growth of AuNRs. The obtained gold nanorods solution was centrifugated twice at 8000 rpm for 15 min with the supernatant solution discarded to remove excess CTAB, some small spherical particles. The AuNRs were then re-suspended in 0.005 M CTAB for storage or re-dispersed in pure water before use. 2.4. Colorimetric detection of endotoxin The sample was prepared as follows. LPS was used in this experiment as prototypical example of endotoxin. LPS solutions
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with different concentrations were prepared in 10 mM phosphate buffer (pH¼7.2) in silanized containers, since it can bind to plastics and certain types of glass. AuNRs with different aspect ratios (AR ¼2.7, and AR ¼3.3) were employed as a comparison in determination of endotoxin. Then 0.1 mL of above LPS solutions (concentration from 0.1 μM to 10 μM) were taken and added into 0.5 mL of the two kinds of AuNRs suspension solutions with appropriate concentration. It should be noticed that the samples were vortexed thoroughly for 5 min to disperse the endotoxin accumulation and guarantee fully interaction before analysis. The UV–vis absorption intensities of the solution were recorded over a range from λ ¼400 nm to λ ¼ 1000 nm, while the color change of the mixture was taken down by a digital camera.
2.5. Pretreatments and analysis of real samples In real sample determinations, milk, carbonic acid drinks purchased from the market and glucose injection purchased from drug store were spiked with endotoxin at different concentration levels. Milk samples should be pretreated as described, with a few modifications in order to remove protein and fat (Li et al., 2010; Kuang et al., 2011). Typically, 1.2 mL of 300 g/L trichloroacetic acid was added into 3.0 mL of the spiked milk samples in a centrifuge tube. After thorough vortex, the mixtures were centrifuged at 10,000 rpm for 10 min, and the supernatant was adjusted to the original volume with trichloroacetic acid again. Finally, the solution was filtered using a syringe and 0.22 μm filter and then used for endotoxin determination. The concentration of endotoxin contained in the samples was quantified by the absorption ratio.
3. Result and discussion 3.1. Colorimetric sensing strategy To better understand the sensing strategy employed in this study, a schematic diagram for the detection of endotoxin using stripped gold nanorods is outlined in Scheme 1. AuNRs prepared are capped with at least a bilayer of CTAB. The surfactant makes AuNRs stable and soluble in aqueous solution and positively charged due to the tetramethylammonium ion (NR4 þ ) as previously studied by El-Sayed (Nikoobakht and El-Sayed, 2001) and Murphy's group (Gole et al., 2004). The LPS bears a net negative charge with the carboxylic groups and phosphate groups in its structure (Scheme 1A). The strong electronegativity and great negative charge density was proved by the zeta potential tested by Sun et al. (2012). Therefore, after adding external endotoxin to the initial AuNRs solution, the attraction of particles with opposite charges leads to random clustering of the two substances. Changes in local surface plasmon resonance directly result in distinguishable color variations of the AuNRs suspensions. For the green AuNRs, the color changes to dark gray gradually (Scheme 1B), and in the case of the red AuNRs, the color shows a change from ruby red to violet in the presence of different concentrations of endotoxin (figure not shown). These changes are easily readout by naked eyes which is the basis of qualitative identification and semi-quantitative analysis of endotoxin. Apart from this, subtle differences can be measured by spectrophotometer for accurate determination. Based on this principle, a simple and rapid determination of endotoxin is developed to trace the smallest amounts of endotoxin in food and injections. Compared to probes constructed by synthesizing and modifying gold nanoparticles, stripped gold nanorods sensor need less steps to prepare and shows multiple performance in the color change and spectrum.
Scheme 1. (A) Structure of lipopolysaccharide; (B) Schematic representations of interaction between endotoxin and gold nanorods.
3.2. Preparation and characterization of AuNRs For the chemical analysis, the discrepancy of AuNRs with different aspect ratios was rarely studied before. In order to reveal the performance of AuNRs aspect ratio in the endotoxin sensing, two types of AuNRs, corresponding to green and red respectively, are chosen among different AuNRs acquired for the further experiments. The two types of AuNRs were fabricated following the same method and procedures and the aspect ratios were tailored by adjusting the addition of AgNO3. The spectral line of the green gold nanorods exhibits a transverse plasmon band at 513 nm and a longitudinal band at 660 nm while the red ones possess two SPR absorption peaks located at 517 nm and 750 nm (Fig. S1A) respectively. These results indicate that AuNRs transverse plasmon band stays almost the same, whereas the longitudinal band varies sharply, which is consistent with the research before (Nikoobakht and El-Sayed, 2003). Transmission electron microscopy provides an opportunity for careful observations of AuNRs. Fig. S1 B and C show the typical TEM images of randomly dispersed green AuNRs and red AuNRs with uniform shapes and sizes. By measuring the diameter and the length of 100 particles in multiple TEM images of the green nanorods and red ones, the aspect ratios are calculated to be 2.7 and 3.3 respectively. Both the statistics and TEM images show that the green rods tend to be short and thick while red ones are slim and slender. In view of that, we reason that the diverse of the charge numbers and their distributions on the surfaces between the green (short) and red (long) rods could possibly lead to the discrepancy of sensitivity in detecting endotoxin. Therefore, two types of rods are involved in subsequent experiments of endotoxin sensing. 3.3. Colorimetric sensing of endotoxin To further verify the mechanism of detecting endotoxin, color changes of the solutions along with the absorption spectra were
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Fig. 1. (A) Photo of the green AuNRs at different endotoxin concentrations; (B) Photo of the red AuNRs at different endotoxin concentrations; (C) UV–vis spectra of the green AuNRs at different endotoxin concentrations; (D) UV–vis spectra of the green AuNRs at different endotoxin concentrations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
recorded. Fig. 1 A and B show photographs corresponding to the green and red AuNRs samples with endotoxin concentrations of 0–10 μM and 0–15 μM respectively. Initially, the color of the short AuNRs suspension is green due to its strong surface plasmon resonance absorption. After addition of the target endotoxin, AuNRs are accumulated, resulting in the color of the colloid change from green to gray. Color change from ruby red to violet takes place for the long rods as well. These changes are related to a plasmon coupling effect of AuNRs: the reduction of the distance between AuNRs particles because of aggregation, leading to a strong enhancement of the localized electric field and increasing refractive indices (Chen et al., 2013; Stewart et al., 2008). Moreover, the phenomenon of aggregation of AuNRs is further evidenced by UV–vis spectra shown in Fig. 1C and D. As expected, with the increase of endotoxin concentration, the absorbance of AuNRs gradually decreases. It is considered that the change of plasmon resonances absorption is related to variation of the concentration of gold nanorods in the suspensions. Addition of endotoxin induces serious aggregation of gold nanorods, leading to the decrease of effective concentration of AuNRs in the solution. Although the two types of rods present similar performance of color change and absorption decrease in the sensing of endotoxin, it should be noticed that there are some different aspects between them. For the green nanorods, as shown in Fig. 1C, a clear red-shift in longitudinal peaks together with significant broadening of the band is observed. On the other hand, the red-shift of longitudinal peaks of the red rods is not obvious but the shift of transverse peaks is more clearly. Apart from this, the ranges for the color change of the two kinds of rods are not the same. The red AuNRs exhibit extension detection range in comparison to that of the green ones. All these results illustrate not only that AuNRs can be used to detect endotoxin, but also AuNRs with different aspect ratios show difference in detection range and sensitivity. More optimizations and comparisons are necessary before the best assay is determined.
3.4. Optimization of experimental conditions In this study, the effect of detection conditions (pH and reaction time between AuNRs and endotoxin) on the experimental results is explored. As a key factor for most electrostatic reactions, the influence caused by pH of the gold nanorods suspension is tested. Generally, CTAB, the typical quaternary ammonium salts, carrying positive charges tend to stay stable in acidic solutions. Previous studies also reveal that higher pH (4 5) weakened CTAB protection against aggregation (Liu et al., 2011). On account of this, the effect of pH on the response of the assay is examined in the range of pH 1–5. In this test, the absorption ratio of longitudinal plasmon band and transverse band is monitored to represent aggregation level. We can see that in Fig. 2 A and B, the absorption ratios of both two types of AuNRs (A660/A513 and A750/A517) reach bottom at pH 2, indicating the maximum aggregation level of gold nanorods. Therefore, pH 2 is set as the operational pH for subsequent experiments. In addition, it should be noticed that the red nanorods show insensitivity to the pH variations in comparison to the green ones, allowing them broad application prospects. Except for pH, reaction time fixing is of vital importance to the chemical process, which is benefit to guarantee reliability of the results and improve detection sensitivity. By recording the signal of absorbance spectra within 30 min, the aggregation kinetic curves of AuNRs at different concentrations (0.1 μM, 0.2 μM and 0.3 μM) of endotoxin are achieved. Fig. 2C and D show that the absorption ratios (A660/A513 and A750/A517) experience a sharp decrease in the first 5 min, but the trend become weaker smoothly afterwards, indicating the slowdown of the aggregating speed. In this case, the colorimetric detection was carried out at the point of fifth-minute after mixing. The same with the pH test, there is little difference in optimal reaction time between the two types of nanorods, suggesting that aggregating rates are similar. After that, colorimetric determination and the spectra absorption are performed at optimized experiment conditions. UV–vis absorption spectra of AuNRs in the presence of endotoxin with
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Fig. 2. (A) Effect of pH value on the absorption ratio (A660/A513) of green AuNRs; (B) Effect of pH value on the absorption ratio (A750/A517) of red AuNRs; (C) Effect of reaction time on the absorption ratio (A660/A513) of green AuNRs with endotoxin concentration of 0.1 μM, 0.2 μM and 0.3 μM; (D) Effect of reaction time on the absorption ratio (A750/A517) of red AuNRs with endotoxin concentration of 0.1 μM, 0.2 μM and 0.3 μM.
Fig. 3. Detection of endotoxin under optimal conditions. (A) Visual color change of the green AuNRs at endotoxin concentration ranges from 0–0.6 μM; (B) Visual color change of the red AuNRs at endotoxin concentration ranges from 0–0.8 μM; (C) UV–vis spectra of the green AuNRs and the plots of A660/A513 versus different endotoxin concentrations corresponding to (A); (D) UV–vis spectra of the red AuNRs and the plots of A750/A517 versus different endotoxin concentrations corresponding to (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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new range of different concentrations were recorded. Fig. 3 A and B illustrate that both the green and red nanorods exhibit a whole color change with endotoxin concentration less than 1 μM. In comparison to the experiment phenomena above, it can be seen that the AuNRs solutions finally turned colorless with high concentration of endotoxin (0.6 μM for the green nanorods). That is because almost all AuNRs are coagulated with endotoxin. When the concentration of endotoxin reaches 0.1 μM under optimal condition, the color change can be evaluated visually, which is much lower than that before optimization. Meanwhile, the variation tendency of spectra curves observed in Fig. 3C and D are similar to the results above. The decrease and the broadening of longitudinal band reflect the aggregation of gold nanorods owing to the electrostatic attraction. However, a slight red-shift in the transverse absorption together with an obvious red-shift of 10 nm in longitudinal absorption peak is observed, indicating shape and size changes of aggregated AuNRs. Furthermore, to assess the accuracy of the assay, a typical plot of the absorption ratios (A660/A513 and A750/A517) in the absence and presence of different concentrations of endotoxin is obtained. A good linear correlation exists between the absorption ratios of AuNRs and the concentration of endotoxin in the range of 0.05–0.4 μM (R ¼0.9977, RSD ¼1.38%) for the green AuNRs and 0.01–0.6 μM (R¼0.986, RSD ¼2.27%) for the red ones (inset of Fig. 3C and D), with the limits of detection to be 0.0096 μM and 0.0084 μM respectively. As mentioned above, red AuNRs have a relatively wider detection range for endotoxin, which is verified in Fig. 3. Nevertheless, the slope of the calibration curve of the red AuNRs is higher than the green ones, showing that red ones exhibit higher sensitivity under current detecting conditions. In this way, the method offers an easy and rapid way for qualitative and semi-quantitative determination of endotoxin. To obtain direct insight into the aggregation of AuNRs and shed light on the detection mechanism, TEM observations of AuNRs with different concentrations of endotoxin are performed. First, the initial AuNRs are uniform and well-dispersible (Fig. S2 A and D). However, after adding 0.1 μM of endotoxin, the random agglomerate of AuNRs, driven by attraction between the positive charges on the surface of AuNRs and negative charges on the endotoxin molecules, is observed (Fig. S2 B and E). When the addition concentration of endotoxin increases up to 0.4 μM, large numbers of rods accumulate (Fig. S2C and F). These TEM images, together with the visible absorption spectra, provide convincing evidence to support our theory of aggregation. Comparison results of the two types of gold rods with different ARs real that the long rods (red AuNRs) show higher crowding level than short ones (green AuNRs) at both endotoxin concentrations (0.1 μM and 0.4 μM), which are consistent with the results of absorption spectra. 3.5. Specificity of the assay Specificity is an important aspect to evaluate the performance of a new proposed assay. Colorimetry based on unmodified AuNRs is commonly designed under critical conditions to avoid interference from other analytes, since it is easily affected by variation of the sensing system. Thus, it is necessary to explore the selectivity of the proposed assay. As mentioned above, AuNRs with higher aspect ratio (longer nanorods) show better sensitivity in detecting endotoxin. Therefore, red (long) nanorods were employed to the interference studies. The specificity is estimated by monitoring the longitude absorbance decrease (Δ A750) in the presence of various bioanalytes in comparison with blank test (mere AuNRs). The results are summarized in Fig. 4. Firstly, the responses of AuNRs to some common inorganic salts, especially phosphates,
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Fig. 4. The corresponding absorbance decrease (Δ A750) of AuNRs with different analytes in comparison to AuNRs solution.
must be tested, since phosphate groups in endotoxin are mainly responsible for the electronegativity of the molecules. The absorbance decrease (Δ A750) shows little change in the presence of 1 mM (100 times higher than endotoxin concentration) of NaCl, Na2CO3, NaH2PO4, and K2HPO4, indicating that their interference is insignificant. Subsequently, as major nutrients in most food, organic acid, small molecule carbohydrate, and amino acid, such as citric acid, ascorbic acid, glucose, sucrose, glutamic acid (Glu) and aspartic acid (ASP) were evaluated. It can be seen that the response of AuNRs to 1 mM of these substances are not comparable to that of endotoxin. Furthermore, some macromolecules, especially those negatively charged, including polysaccharides and proteins, were evaluated. 0.1 mM of chitosan (50 kDa), beta-glucan (20 kDa), methylcellulose (14 kDa), bovine serum albumin (BSA, 66.4 kDa), and ovalbumin (OVA, 44 kDa) were detected by the present method. Among these chemicals, methylcellulose results in an absorbance decrease to a certain degree. That is possibly due to the viscosity, which could promote assembly of gold nanorods. However, the absorbance decrease (Δ A750) caused by methylcellulose is negligible when compare to that of endotoxin. At last, compounds with hydrophobic tails were tested in order to further verify the aggregation mechanism. 1 mM dodecyl trimethyl ammonium bromide (DTAB), Tween-20 and Polyvinyl alcohol (PVA, 160 kDa) show little influence on AuNRs. Interestingly, after adding 1 mM phosphatidylchline into the AuNRs, an absorption decrease is observed. This is due to the negatively charged phosphate group in the molecule, which is similar to the endotoxin. However, the electrostatic attraction between endotoxin and AuNRs is remarkably stronger than phosphatidylchline since it has more phosphate groups and carboxylic groups which give it greater negative charge density. Nevertheless, in all cases, no color change of the gold nanorods suspensions is observed in addition of the substances, implying that the assay proposed is of good specificity and has application potential in real samples. 3.6. Analysis of endotoxin in real samples Food and drinks can be contaminated by bacteria in any step of the industry chain of cultivation and breeding, producing, processing, packaging and selling. When the bacteria are destroyed owing to secondary processing before appearance on a dinner table, endotoxin can be released and become a potential hazard. Moreover, determination of endotoxin in medical apparatus and injections is a routine in all intravenous therapy. Having achieved satisfactory sensitivity and specificity in standard solutions, the feasibility of the endotoxin sensor was tested through interaction
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Table 1 Detection results of endotoxin levels in spiked milk, sprit and glucose injections. Samples
Added concentration (μM)
Measured concentration (μM)
Recovery (%)
CV (%)
Milk 1 Milk 2 Glc injection 1 Glc injection 2 Sprite 1 Sprite 2
0.01 0.05 0.1 0.2 0.3 0.4
0.0086 0.0424 0.0988 0.2168 0.2837 0.3874
86 84.8 98.81 108.4 94.46 96.85
9.3512 7.3479 6.4234 7.2352 4.3982 8.64
Science Foundation of China (Nos. 31101274, 31201357). Authors also thank the financial support from Shaanxi Province for New Stars of Provincial Young Scientific Worker (2012KJXX-17).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.12.006.
References with unbroken bacteria cells in previous work by Sun et al. (2012). In this study, the assay is expected to be applied to detect endotoxin residues after processing in real samples. Therefore, to explore whether the detection is influenced by matrices in practical samples, milk, sprits, and glucose injections were chosen for the analysis. First, it is found that no endotoxin was detected in the original samples by the present approach. Then, a standard addition method for analyzing endotoxin in real samples was adopted in this study. Generally, the samples were spiked and treated according to the procedure described in Section 2, and the results are listed in Table 1. It can be seen that the gold nanorods exhibit a good linear correlation and a sensitive response to the change of different concentrations of endotoxin from 0.01 μM to 0.4 μM. The recoveries of spiked samples range from 84% to 109% with the coefficient of variation less than 10% (n ¼ 4), indicating the promising feasibility of this colorimetry for endotoxin quantification. Furthermore, when the spiked concentration is higher than 0.2 μM, the colors of the measured samples show evident changes. This phenomenon is well agreed with the standard solutions described above. The direct and rapid detection of endotoxin provided is highly preferred in the supervisory of food contaminations by microorganism. 4. Conclusion In summary, we report an AuNRs-based probe for the colorimetric and selective detection of endotoxin by counterionmediated aggregations. This method is rapid and convenient, which can be accomplished within 15 min based on the variation of UV–vis spectra and a significant color change that is easily recognized by naked eyes. Parameters that affect the sensitivity and the possible interferential substances of the experiment were investigated. The proposed assay presents satisfactory linear range, low detection limit, good accuracy and specificity in the detection of endotoxin. In particular, we also probed into discrepancy of detecting sensitivity and linear interval by AuNRs with different aspect ratios. Results indicate that AuNRs with relative higher aspect ratio (longer ones) show advantages. Furthermore, we have demonstrated that our method is suitable to monitor the amount of endotoxin in food and injection samples efficiently, which can be applied as a promising candidate for on-site detection of endotoxin. Acknowledgments This work is supported by National Science & Technology Pillar Program (2012BAK17B06, 2012BAH30F03), National Natural
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Facile colorimetric method for simple and rapid detection of endotoxin based on counterion-mediated gold nanorods aggregation Yashan Wang 1, Daohong Zhang 1, Wei Liu, Xiao Zhang, Shaoxuan Yu, Tao Liu, Wentao Zhang, Wenxin Zhu, Jianlong Wang n College of Food Science and Engineering, Northwest A&F University, Yangling 712100, Shaanxi, China
art ic l e i nf o
a b s t r a c t
Article history: Received 13 September 2013 Received in revised form 18 November 2013 Accepted 2 December 2013 Available online 11 December 2013
Existence of endotoxin in food and injection products indicates bacterial contaminations and therefore poses threat to human health. Herein, a simple and rapid colorimetric method for the effective detection of endotoxin in food and injections based on counterion-mediated gold nanorods aggregation is first proposed. By taking advantage of the color change of unmodified gold nanorods resulted from endotoxin mediated gold nanorods aggregation, endotoxin could be detected in the concentration range of 0.01–0.6 μM. Further, we studied the performance of gold nanorods with different aspect ratios (2.7 and 3.3) in determination of endotoxin and found that gold nanorods with higher aspect ratio (AR) showed superiority in the sensing sensitivity of endotoxin. A good specificity for endotoxin, a detection limit of 0.0084 μM and recoveries ranging from 84% to 109% in spiked food and injection samples are obtained with the colorimetric method. Results demonstrate that the present method provides a novel and effective approach for on-site screening of endotoxin in common products, which is beneficial for monitoring and reducing the risk of bacterial contaminations in food and injections production. & 2013 Elsevier B.V. All rights reserved.
Keywords: Endotoxin Gold nanorods Colorimetric detection
1. Introduction Endotoxin, covering the adhesion peptide of the Gram-negative bacteria outer membrane, is a significant contaminant in both food industry and pharmaceutical production (Kim et al., 2006). The prototypical example of endotoxin in most bacteria is lipopolysaccharide (LPS), which is in fact the hazard of endotoxin. LPS consists of a large polysaccharide chain of repeating oligosaccharide subunits, a core oligosaccharide and a unique structure of lipid moiety, known as lipid A, which is mainly responsible for the biological toxicity, causing various effects on human, including fever, diarrhea, vomiting, septic shock, inflammatory reaction, low blood pressure and disseminated intravascular coagulation (Kim et al., 2006; Limbut et al., 2007). Septic shock induced by endotoxin is a significant threat to public health with more than 150 thousand related infection cases reported every year in the United States alone. Worldwide, the situation is not optimistic, especially in areas without sufficient sterilization and water treatment facilities (Uzarski and Mello, 2012). Large amounts of endotoxin can be mobilized only when the bacteria dissolved due to aqtocytolysis or artificially damaged (Cho et al., 2012), as a
n
Corresponding author. Tel.: þ 86 29 8709 2275; fax: þ 86 29 8709 2275. E-mail address: [email protected] (J. Wang). 1 Both of Yashan Wang and Daohong Zhang rank the first authors.
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.006
result, massive existence of endotoxin in water or drugs implies severe bacterial contamination. Although endotoxin is released after death of bacterial cells, it can survive continuously heating for 1 h at 100 1C. Therefore, it is intractable to control and remove in both food and drug manufacture procedures and environmental disinfection. On account of this, it is necessary to detect and remove endotoxin in food and drug production process as well as the final merchandise, as even small amounts of endotoxin will pose a serious threat to human health. Thus, various approaches are put forward for this purpose. Endotoxin is commonly quantified via the Limulus Amebocyte Lysate (LAL) assay based on an enzymatic coagulation of the blood of a primitive marine arthropod, the horseshoe crab, in the presence of endotoxin. This method, while sensitive, suffers from environmental sensitivity (pyrogen in water, pH and temperature fluctuation) as well as false positive responses to other carbohydrate-like molecules, such as β-glucan (Uzarski and Mello, 2012; US FDA, 1987). Besides, it has limited ability to detect inactive endotoxin in the system (Cheng et al., 2012). More importantly, LAL is extracted from the horseshoe crab, which is on the brink of extinction, and authorities have promulgated laws to protect them from overwhelming fishing (Sun et al., 2012). To break the ice, several new analytical techniques have been reported. A colorimetric endotoxin sensor prepared by functionalized polydiacetylene liposomes to provide differential responses to specific LPS strains was reported (Rangin and Basu, 2004). Then,
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fluorescently labeled analogs of two peptide variants derived from the LPS-binding protein CD14 were developed to detect and discriminate endotoxin (Voss et al., 2007). In addition, antimicrobial peptides, together with surface plasmon resonance were employed to detect and classify different lipopolysaccharide molecules (Uzarski and Mello, 2012). Besides, the improved SDS-PAGE technique is also available to detect LPS sensitively (Redwan, 2012). An innovatively designed method to detect endotoxin taking advantage of cysteamine-modified gold nanoparticles was proposed. It was the first attempt to determine endotoxin by nanostructure of noble metal which received a detection limit as low as 0.33 nM and applied to recognize the surface of Escherichia coli (Sun et al., 2012). Except for this, an electrochemical detection of LPS using a combination of ferrocene-attached polymyxin B and a modified glassy carbon electrode (Iijima et al., 2011) was carried out, after which, amine-terminated aptamer immobilized gold electrode was formed as an electrochemical endotoxin specific biosensor (Su et al., 2012). However, these methods are either expensive, labor intensive or time consuming, making them unsuitable for on-site analysis. Therefore, it is imperative to develop an easy-handled, cost-effective and environmentallyfriendly approach for endotoxin on-spot detection. In the past decades, gold nanorods (AuNRs) have been employed for a range of applications, including biosensing (Yu and Irudayaraj, 2007; He et al., 2008), cell imaging (Wang et al., 2009), medical diagnostics (Truong et al., 2011), and cancer photothermal therapy (Wang et al., 2011). AuNRs have been proved to be versatile and tunable materials compared to other materials including spherical nanoparticles, especially in the detection field due to the shape anisotropy of nanorods. They possess two directional electron oscillations in response to the polarization of the incident light, the transverse plasmon band and the longitudinal plasmon band, corresponding to light absorption and scattering along the short axis of the nanorods, and the long axis respectively. Particularly, the longitudinal absorption band is extremely sensitive to the changes in the dielectric properties of the surroundings, such as solvents, adsorbates, and the interparticle distance of the gold nanorods owing to localized surface plasmon resonance (LSPR) (Ho et al., 2012; Chen et al., 2013). Nevertheless, an absorption coefficient of the peak at the longitudinal plasmon band is quite large and this absorption decreases remarkably upon aggregation (Kitazaki et al., 2012), and even, in some cases, a new red-shifted absorption band would occur gradually. At the same time, a change of the color of AuNRs suspension can be observed due to the shift of absorption band. These properties suggest gold nanorods could enable highly sensitive, specific and visualized detection of biomolecules based on the aggregation mechanism (Niidome et al., 2010). For this reason, gold nanorods, modified or unmodified, as a powerful tool have been utilized to detect and monitor diverse targets, including amino acids (Sudeep et al., 2005; Wang et al., 2012), proteins (Zhu et al., 2011; Xu et al., 2012; Yasun et al., 2012; Zhang and Shen, 2013), multiple pathogens (Wang and Irudayaraj, 2008), different metal ions (Huang et al., 2013; Wang et al., 2013; Liang et al., 2012; Anand et al., 2013), disease markers (Truong et al., 2011; Huang et al., 2012) and some other chemical substances. However, to the best of our knowledge, strategy based on the aggregation of gold nanorods has not been reported for the selective sensing of endotoxin. Herein, for the sake of reducing microbial contaminants and toxins in food, decreasing harm caused by bacteria to humanity, we report a method for rapid and visualized detection of endotoxin in food and injections taking advantage of the counterion electrostatic interaction between endotoxin and unmodified gold nanorods. Furthermore, we explored the performance of gold nanorods with different aspect ratios (the ratio between the length and diameter of the rods) in sensitivity of quantitative
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determination. And the selectivity of this assay was also investigated. Finally, to validate the feasibility of the proposed method, several beverages, including milk, carbonic acid drink, and glucose injection were chosen to test the accuracy of measurements. We thus performed a rapid and reliable assay for the detection of the endotoxin level in food and drugs, and providing a possibility for improving the current situation of food and drugs contamination by bacteria.
2. Experiment 2.1. Chemicals and materials Chloroauric acid (HAuCl4 3H2O) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Silver nitrate (AgNO3), Cetyltrimethylammonium bromide (CTAB), and Lipopolysaccharide (LPS), which is extracted from E. Coli 055:B5, was purchased from Sigma (St. Louis, USA). The LPS molecular weight was measured to be 10 kDa in previous work (Sun et al., 2012). All reagents were at least of analytical reagent grade and used as received. Ultrapure water (18.2 MΩ cm), obtained from a water purification system, was used in the whole experiment. All the glassware was cleaned with aqua regia and thoroughly rinsed with ultrapure water before use. 2.2. Instrumentation The absorption spectra were recorded on a UV-2550 spectrophotometer (Shimadzu, Japan) with 1 cm pathlength cells at room temperature. Transmission electron microscopy (TEM) measurements were performed on a HT7700 (Hitachi, Japan) at 80 kV. 2.3. Synthesis of gold nanorods AuNRs were fabricated following the silver ion-assisted seedmediated and CTAB surfactant-directed method according to previous report (Nikoobakht and El-Sayed, 2003; Fu et al., 2012) with necessary modifications. Typically, the seed solution was prepared by the addition of HAuCl4 (0.6 mM, 2.5 mL) into CTAB (0.2 M, 2.5 mL) with gentle mixing. Subsequently, to the mixture solution, freshly prepared ice-cold NaBH4 solution (0.01 M, 0.3 mL) was added quickly, and the color of the solution changed from dark yellow to brown under vigorous stirring for 2 min. The obtained solution was kept as the seed solution and used within 4 h. Gold nanorods were prepared using an aqueous growth solution of CTAB (0.1 M, 40 mL) in a 50 mL plastic tube. To this solution, HAuCl4 (50 mM, 0.6 mL) and AgNO3 (50 mM, 0.03– 0.15 mL) were added with gentle inversion. The addition of different volumes of AgNO3 produces gold nanorods with various aspect ratios (Nikoobakht and El-Sayed, 2003). Then 0.5 mL of 0.08 M ascorbic acid was added, followed by a color change of the solution from dark yellow to colorless. Finally, 0.8 mL of seed solution was added to the growth solution and gently shaken for 10 s. The solution was left undisturbed overnight at 28 1C to ensure fully growth of AuNRs. The obtained gold nanorods solution was centrifugated twice at 8000 rpm for 15 min with the supernatant solution discarded to remove excess CTAB, some small spherical particles. The AuNRs were then re-suspended in 0.005 M CTAB for storage or re-dispersed in pure water before use. 2.4. Colorimetric detection of endotoxin The sample was prepared as follows. LPS was used in this experiment as prototypical example of endotoxin. LPS solutions
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with different concentrations were prepared in 10 mM phosphate buffer (pH¼7.2) in silanized containers, since it can bind to plastics and certain types of glass. AuNRs with different aspect ratios (AR ¼2.7, and AR ¼3.3) were employed as a comparison in determination of endotoxin. Then 0.1 mL of above LPS solutions (concentration from 0.1 μM to 10 μM) were taken and added into 0.5 mL of the two kinds of AuNRs suspension solutions with appropriate concentration. It should be noticed that the samples were vortexed thoroughly for 5 min to disperse the endotoxin accumulation and guarantee fully interaction before analysis. The UV–vis absorption intensities of the solution were recorded over a range from λ ¼400 nm to λ ¼ 1000 nm, while the color change of the mixture was taken down by a digital camera.
2.5. Pretreatments and analysis of real samples In real sample determinations, milk, carbonic acid drinks purchased from the market and glucose injection purchased from drug store were spiked with endotoxin at different concentration levels. Milk samples should be pretreated as described, with a few modifications in order to remove protein and fat (Li et al., 2010; Kuang et al., 2011). Typically, 1.2 mL of 300 g/L trichloroacetic acid was added into 3.0 mL of the spiked milk samples in a centrifuge tube. After thorough vortex, the mixtures were centrifuged at 10,000 rpm for 10 min, and the supernatant was adjusted to the original volume with trichloroacetic acid again. Finally, the solution was filtered using a syringe and 0.22 μm filter and then used for endotoxin determination. The concentration of endotoxin contained in the samples was quantified by the absorption ratio.
3. Result and discussion 3.1. Colorimetric sensing strategy To better understand the sensing strategy employed in this study, a schematic diagram for the detection of endotoxin using stripped gold nanorods is outlined in Scheme 1. AuNRs prepared are capped with at least a bilayer of CTAB. The surfactant makes AuNRs stable and soluble in aqueous solution and positively charged due to the tetramethylammonium ion (NR4 þ ) as previously studied by El-Sayed (Nikoobakht and El-Sayed, 2001) and Murphy's group (Gole et al., 2004). The LPS bears a net negative charge with the carboxylic groups and phosphate groups in its structure (Scheme 1A). The strong electronegativity and great negative charge density was proved by the zeta potential tested by Sun et al. (2012). Therefore, after adding external endotoxin to the initial AuNRs solution, the attraction of particles with opposite charges leads to random clustering of the two substances. Changes in local surface plasmon resonance directly result in distinguishable color variations of the AuNRs suspensions. For the green AuNRs, the color changes to dark gray gradually (Scheme 1B), and in the case of the red AuNRs, the color shows a change from ruby red to violet in the presence of different concentrations of endotoxin (figure not shown). These changes are easily readout by naked eyes which is the basis of qualitative identification and semi-quantitative analysis of endotoxin. Apart from this, subtle differences can be measured by spectrophotometer for accurate determination. Based on this principle, a simple and rapid determination of endotoxin is developed to trace the smallest amounts of endotoxin in food and injections. Compared to probes constructed by synthesizing and modifying gold nanoparticles, stripped gold nanorods sensor need less steps to prepare and shows multiple performance in the color change and spectrum.
Scheme 1. (A) Structure of lipopolysaccharide; (B) Schematic representations of interaction between endotoxin and gold nanorods.
3.2. Preparation and characterization of AuNRs For the chemical analysis, the discrepancy of AuNRs with different aspect ratios was rarely studied before. In order to reveal the performance of AuNRs aspect ratio in the endotoxin sensing, two types of AuNRs, corresponding to green and red respectively, are chosen among different AuNRs acquired for the further experiments. The two types of AuNRs were fabricated following the same method and procedures and the aspect ratios were tailored by adjusting the addition of AgNO3. The spectral line of the green gold nanorods exhibits a transverse plasmon band at 513 nm and a longitudinal band at 660 nm while the red ones possess two SPR absorption peaks located at 517 nm and 750 nm (Fig. S1A) respectively. These results indicate that AuNRs transverse plasmon band stays almost the same, whereas the longitudinal band varies sharply, which is consistent with the research before (Nikoobakht and El-Sayed, 2003). Transmission electron microscopy provides an opportunity for careful observations of AuNRs. Fig. S1 B and C show the typical TEM images of randomly dispersed green AuNRs and red AuNRs with uniform shapes and sizes. By measuring the diameter and the length of 100 particles in multiple TEM images of the green nanorods and red ones, the aspect ratios are calculated to be 2.7 and 3.3 respectively. Both the statistics and TEM images show that the green rods tend to be short and thick while red ones are slim and slender. In view of that, we reason that the diverse of the charge numbers and their distributions on the surfaces between the green (short) and red (long) rods could possibly lead to the discrepancy of sensitivity in detecting endotoxin. Therefore, two types of rods are involved in subsequent experiments of endotoxin sensing. 3.3. Colorimetric sensing of endotoxin To further verify the mechanism of detecting endotoxin, color changes of the solutions along with the absorption spectra were
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Fig. 1. (A) Photo of the green AuNRs at different endotoxin concentrations; (B) Photo of the red AuNRs at different endotoxin concentrations; (C) UV–vis spectra of the green AuNRs at different endotoxin concentrations; (D) UV–vis spectra of the green AuNRs at different endotoxin concentrations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
recorded. Fig. 1 A and B show photographs corresponding to the green and red AuNRs samples with endotoxin concentrations of 0–10 μM and 0–15 μM respectively. Initially, the color of the short AuNRs suspension is green due to its strong surface plasmon resonance absorption. After addition of the target endotoxin, AuNRs are accumulated, resulting in the color of the colloid change from green to gray. Color change from ruby red to violet takes place for the long rods as well. These changes are related to a plasmon coupling effect of AuNRs: the reduction of the distance between AuNRs particles because of aggregation, leading to a strong enhancement of the localized electric field and increasing refractive indices (Chen et al., 2013; Stewart et al., 2008). Moreover, the phenomenon of aggregation of AuNRs is further evidenced by UV–vis spectra shown in Fig. 1C and D. As expected, with the increase of endotoxin concentration, the absorbance of AuNRs gradually decreases. It is considered that the change of plasmon resonances absorption is related to variation of the concentration of gold nanorods in the suspensions. Addition of endotoxin induces serious aggregation of gold nanorods, leading to the decrease of effective concentration of AuNRs in the solution. Although the two types of rods present similar performance of color change and absorption decrease in the sensing of endotoxin, it should be noticed that there are some different aspects between them. For the green nanorods, as shown in Fig. 1C, a clear red-shift in longitudinal peaks together with significant broadening of the band is observed. On the other hand, the red-shift of longitudinal peaks of the red rods is not obvious but the shift of transverse peaks is more clearly. Apart from this, the ranges for the color change of the two kinds of rods are not the same. The red AuNRs exhibit extension detection range in comparison to that of the green ones. All these results illustrate not only that AuNRs can be used to detect endotoxin, but also AuNRs with different aspect ratios show difference in detection range and sensitivity. More optimizations and comparisons are necessary before the best assay is determined.
3.4. Optimization of experimental conditions In this study, the effect of detection conditions (pH and reaction time between AuNRs and endotoxin) on the experimental results is explored. As a key factor for most electrostatic reactions, the influence caused by pH of the gold nanorods suspension is tested. Generally, CTAB, the typical quaternary ammonium salts, carrying positive charges tend to stay stable in acidic solutions. Previous studies also reveal that higher pH (4 5) weakened CTAB protection against aggregation (Liu et al., 2011). On account of this, the effect of pH on the response of the assay is examined in the range of pH 1–5. In this test, the absorption ratio of longitudinal plasmon band and transverse band is monitored to represent aggregation level. We can see that in Fig. 2 A and B, the absorption ratios of both two types of AuNRs (A660/A513 and A750/A517) reach bottom at pH 2, indicating the maximum aggregation level of gold nanorods. Therefore, pH 2 is set as the operational pH for subsequent experiments. In addition, it should be noticed that the red nanorods show insensitivity to the pH variations in comparison to the green ones, allowing them broad application prospects. Except for pH, reaction time fixing is of vital importance to the chemical process, which is benefit to guarantee reliability of the results and improve detection sensitivity. By recording the signal of absorbance spectra within 30 min, the aggregation kinetic curves of AuNRs at different concentrations (0.1 μM, 0.2 μM and 0.3 μM) of endotoxin are achieved. Fig. 2C and D show that the absorption ratios (A660/A513 and A750/A517) experience a sharp decrease in the first 5 min, but the trend become weaker smoothly afterwards, indicating the slowdown of the aggregating speed. In this case, the colorimetric detection was carried out at the point of fifth-minute after mixing. The same with the pH test, there is little difference in optimal reaction time between the two types of nanorods, suggesting that aggregating rates are similar. After that, colorimetric determination and the spectra absorption are performed at optimized experiment conditions. UV–vis absorption spectra of AuNRs in the presence of endotoxin with
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Fig. 2. (A) Effect of pH value on the absorption ratio (A660/A513) of green AuNRs; (B) Effect of pH value on the absorption ratio (A750/A517) of red AuNRs; (C) Effect of reaction time on the absorption ratio (A660/A513) of green AuNRs with endotoxin concentration of 0.1 μM, 0.2 μM and 0.3 μM; (D) Effect of reaction time on the absorption ratio (A750/A517) of red AuNRs with endotoxin concentration of 0.1 μM, 0.2 μM and 0.3 μM.
Fig. 3. Detection of endotoxin under optimal conditions. (A) Visual color change of the green AuNRs at endotoxin concentration ranges from 0–0.6 μM; (B) Visual color change of the red AuNRs at endotoxin concentration ranges from 0–0.8 μM; (C) UV–vis spectra of the green AuNRs and the plots of A660/A513 versus different endotoxin concentrations corresponding to (A); (D) UV–vis spectra of the red AuNRs and the plots of A750/A517 versus different endotoxin concentrations corresponding to (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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new range of different concentrations were recorded. Fig. 3 A and B illustrate that both the green and red nanorods exhibit a whole color change with endotoxin concentration less than 1 μM. In comparison to the experiment phenomena above, it can be seen that the AuNRs solutions finally turned colorless with high concentration of endotoxin (0.6 μM for the green nanorods). That is because almost all AuNRs are coagulated with endotoxin. When the concentration of endotoxin reaches 0.1 μM under optimal condition, the color change can be evaluated visually, which is much lower than that before optimization. Meanwhile, the variation tendency of spectra curves observed in Fig. 3C and D are similar to the results above. The decrease and the broadening of longitudinal band reflect the aggregation of gold nanorods owing to the electrostatic attraction. However, a slight red-shift in the transverse absorption together with an obvious red-shift of 10 nm in longitudinal absorption peak is observed, indicating shape and size changes of aggregated AuNRs. Furthermore, to assess the accuracy of the assay, a typical plot of the absorption ratios (A660/A513 and A750/A517) in the absence and presence of different concentrations of endotoxin is obtained. A good linear correlation exists between the absorption ratios of AuNRs and the concentration of endotoxin in the range of 0.05–0.4 μM (R ¼0.9977, RSD ¼1.38%) for the green AuNRs and 0.01–0.6 μM (R¼0.986, RSD ¼2.27%) for the red ones (inset of Fig. 3C and D), with the limits of detection to be 0.0096 μM and 0.0084 μM respectively. As mentioned above, red AuNRs have a relatively wider detection range for endotoxin, which is verified in Fig. 3. Nevertheless, the slope of the calibration curve of the red AuNRs is higher than the green ones, showing that red ones exhibit higher sensitivity under current detecting conditions. In this way, the method offers an easy and rapid way for qualitative and semi-quantitative determination of endotoxin. To obtain direct insight into the aggregation of AuNRs and shed light on the detection mechanism, TEM observations of AuNRs with different concentrations of endotoxin are performed. First, the initial AuNRs are uniform and well-dispersible (Fig. S2 A and D). However, after adding 0.1 μM of endotoxin, the random agglomerate of AuNRs, driven by attraction between the positive charges on the surface of AuNRs and negative charges on the endotoxin molecules, is observed (Fig. S2 B and E). When the addition concentration of endotoxin increases up to 0.4 μM, large numbers of rods accumulate (Fig. S2C and F). These TEM images, together with the visible absorption spectra, provide convincing evidence to support our theory of aggregation. Comparison results of the two types of gold rods with different ARs real that the long rods (red AuNRs) show higher crowding level than short ones (green AuNRs) at both endotoxin concentrations (0.1 μM and 0.4 μM), which are consistent with the results of absorption spectra. 3.5. Specificity of the assay Specificity is an important aspect to evaluate the performance of a new proposed assay. Colorimetry based on unmodified AuNRs is commonly designed under critical conditions to avoid interference from other analytes, since it is easily affected by variation of the sensing system. Thus, it is necessary to explore the selectivity of the proposed assay. As mentioned above, AuNRs with higher aspect ratio (longer nanorods) show better sensitivity in detecting endotoxin. Therefore, red (long) nanorods were employed to the interference studies. The specificity is estimated by monitoring the longitude absorbance decrease (Δ A750) in the presence of various bioanalytes in comparison with blank test (mere AuNRs). The results are summarized in Fig. 4. Firstly, the responses of AuNRs to some common inorganic salts, especially phosphates,
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Fig. 4. The corresponding absorbance decrease (Δ A750) of AuNRs with different analytes in comparison to AuNRs solution.
must be tested, since phosphate groups in endotoxin are mainly responsible for the electronegativity of the molecules. The absorbance decrease (Δ A750) shows little change in the presence of 1 mM (100 times higher than endotoxin concentration) of NaCl, Na2CO3, NaH2PO4, and K2HPO4, indicating that their interference is insignificant. Subsequently, as major nutrients in most food, organic acid, small molecule carbohydrate, and amino acid, such as citric acid, ascorbic acid, glucose, sucrose, glutamic acid (Glu) and aspartic acid (ASP) were evaluated. It can be seen that the response of AuNRs to 1 mM of these substances are not comparable to that of endotoxin. Furthermore, some macromolecules, especially those negatively charged, including polysaccharides and proteins, were evaluated. 0.1 mM of chitosan (50 kDa), beta-glucan (20 kDa), methylcellulose (14 kDa), bovine serum albumin (BSA, 66.4 kDa), and ovalbumin (OVA, 44 kDa) were detected by the present method. Among these chemicals, methylcellulose results in an absorbance decrease to a certain degree. That is possibly due to the viscosity, which could promote assembly of gold nanorods. However, the absorbance decrease (Δ A750) caused by methylcellulose is negligible when compare to that of endotoxin. At last, compounds with hydrophobic tails were tested in order to further verify the aggregation mechanism. 1 mM dodecyl trimethyl ammonium bromide (DTAB), Tween-20 and Polyvinyl alcohol (PVA, 160 kDa) show little influence on AuNRs. Interestingly, after adding 1 mM phosphatidylchline into the AuNRs, an absorption decrease is observed. This is due to the negatively charged phosphate group in the molecule, which is similar to the endotoxin. However, the electrostatic attraction between endotoxin and AuNRs is remarkably stronger than phosphatidylchline since it has more phosphate groups and carboxylic groups which give it greater negative charge density. Nevertheless, in all cases, no color change of the gold nanorods suspensions is observed in addition of the substances, implying that the assay proposed is of good specificity and has application potential in real samples. 3.6. Analysis of endotoxin in real samples Food and drinks can be contaminated by bacteria in any step of the industry chain of cultivation and breeding, producing, processing, packaging and selling. When the bacteria are destroyed owing to secondary processing before appearance on a dinner table, endotoxin can be released and become a potential hazard. Moreover, determination of endotoxin in medical apparatus and injections is a routine in all intravenous therapy. Having achieved satisfactory sensitivity and specificity in standard solutions, the feasibility of the endotoxin sensor was tested through interaction
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Table 1 Detection results of endotoxin levels in spiked milk, sprit and glucose injections. Samples
Added concentration (μM)
Measured concentration (μM)
Recovery (%)
CV (%)
Milk 1 Milk 2 Glc injection 1 Glc injection 2 Sprite 1 Sprite 2
0.01 0.05 0.1 0.2 0.3 0.4
0.0086 0.0424 0.0988 0.2168 0.2837 0.3874
86 84.8 98.81 108.4 94.46 96.85
9.3512 7.3479 6.4234 7.2352 4.3982 8.64
Science Foundation of China (Nos. 31101274, 31201357). Authors also thank the financial support from Shaanxi Province for New Stars of Provincial Young Scientific Worker (2012KJXX-17).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.12.006.
References with unbroken bacteria cells in previous work by Sun et al. (2012). In this study, the assay is expected to be applied to detect endotoxin residues after processing in real samples. Therefore, to explore whether the detection is influenced by matrices in practical samples, milk, sprits, and glucose injections were chosen for the analysis. First, it is found that no endotoxin was detected in the original samples by the present approach. Then, a standard addition method for analyzing endotoxin in real samples was adopted in this study. Generally, the samples were spiked and treated according to the procedure described in Section 2, and the results are listed in Table 1. It can be seen that the gold nanorods exhibit a good linear correlation and a sensitive response to the change of different concentrations of endotoxin from 0.01 μM to 0.4 μM. The recoveries of spiked samples range from 84% to 109% with the coefficient of variation less than 10% (n ¼ 4), indicating the promising feasibility of this colorimetry for endotoxin quantification. Furthermore, when the spiked concentration is higher than 0.2 μM, the colors of the measured samples show evident changes. This phenomenon is well agreed with the standard solutions described above. The direct and rapid detection of endotoxin provided is highly preferred in the supervisory of food contaminations by microorganism. 4. Conclusion In summary, we report an AuNRs-based probe for the colorimetric and selective detection of endotoxin by counterionmediated aggregations. This method is rapid and convenient, which can be accomplished within 15 min based on the variation of UV–vis spectra and a significant color change that is easily recognized by naked eyes. Parameters that affect the sensitivity and the possible interferential substances of the experiment were investigated. The proposed assay presents satisfactory linear range, low detection limit, good accuracy and specificity in the detection of endotoxin. In particular, we also probed into discrepancy of detecting sensitivity and linear interval by AuNRs with different aspect ratios. Results indicate that AuNRs with relative higher aspect ratio (longer ones) show advantages. Furthermore, we have demonstrated that our method is suitable to monitor the amount of endotoxin in food and injection samples efficiently, which can be applied as a promising candidate for on-site detection of endotoxin. Acknowledgments This work is supported by National Science & Technology Pillar Program (2012BAK17B06, 2012BAH30F03), National Natural
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