A simple and specific assay for real-time colorimetric visualization of β-lactamase activity by using gold nanoparticles

DOI: 10.1002/anie.200702773

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Angewandte Chemie - International Edition p. 8799 - 8803 (2007)

Update date:2022-08-02

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Authors:

Liu, Rongrong

Liew, Roushen

Zhou, Jie

Xing, Bengang

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Article abstract of DOI:10.1002/anie.200702773

(Chemical Equation Presented) The color of gold : Bacterial β-lactamases (Blas) can be rapidly identified by a simple and specific colorimetric assay with gold nanoparticles (Au-NPs). This assay can evaluate enzymatic kinetic reactions and screen inhibito

Full text of DOI:10.1002/anie.200702773

Angewandte

Chemie

DOI: 10.1002/anie.200702773

Colorimetric Assays

A Simple and Specific Assay for Real-Time Colorimetric Visualization

of b-Lactamase Activity by Using Gold Nanoparticles**

Rongrong Liu, Roushen Liew, Jie Zhou, and Bengang Xing*

b-Lactamases (Blas) are a family of bacterial enzymes that

can hydrolyze the b-lactam ring in penicillins and cephalo-

sporins with high catalytic efficiency and render the bacteria

resistant to the b-lactam antimicrobial reagents.^[1]Currently,

increased resistance of bacterial infections to antibiotic

treatment has been extensively documented and has

become a generally recognized problem for clinicians world-

sensitive measurement of metal ions,^[10]optical study of a

single virus,^[11]and monitoring special enzyme activities such

as alkaline phosphatases,kinases,proteases,etc. ^[12]Currently,

most of Au-NP-based colorimetric assays for enzyme detec-

tion are mainly dependent on the enzyme properties to induce

the change in gold-particle aggregates. One design to achieve

this target is to monitor the Au-NP agglomeration states from

aggregation to dispersion. In this case,enzymes react with

DNA or peptide-functionalized nanoparticles and induce

their disassembly. The dispersion of Au-NP aggregates leads

to significant color changes. This type of colorimetric assay

has been proved to be simpler,more direct and accurate,and

has been successfully used to sensitively determine the

presence of the enzymes in real time.^[8d,12d]One aspect that

needs to be improved for this assay is the steric hindrance and

slow enzyme kinetics caused by the bulky conjugation of

enzyme substrates with Au-NPs. An alternative design for the

Au-NP-based colorimetric assay relies on the assembly

process of dispersed gold particles. In this detection system,

enzymes react with free substrates first. The released products

are then able to cross-link the Au-NPs,which leads to

aggregates. This type of assay is less sterically hindered,

exhibits fast enzyme kinetics,and is more efficient in

monitoring the enzyme activity without functionalization of

the Au-NPs.^[12a,b]We have developed a novel and easily

operational chromogenic assay based on Au-NP aggregates to

detect Bla activity in vitro and in antibiotic-resistant bacterial

suspensions. This method can be used to rapidly identify the

Bla molecules and screen the enzyme inhibitors in a high-

throughput fashion by the naked eye or a simple colorimetric

reader.

Scheme 1 illustrates the simple procedure of using Au-

NPs to detect Bla. Cleavage of the b-lactam ring in a

cephalosporin triggers spontaneous elimination of any leav-

ing groups previously attached to the 3’-position. Here,two

cephem nuclei are connected through a dithiol-modified 1,2-

bis(2-aminoethoxy)ethane flexible linker after iodo substitu-

tion. The thiol group is an excellent leaving group and will

facilitate fragmentation upon enzyme treatment. Another

advantage of the introduction of a thiol group is that its strong

interactions with gold surfaces lead to the aggregation of gold

nanoparticles. 1,2-Bis(2-aminoethoxy)ethane was used here

to improve the substrate solubility and to minimize the steric

interactions between the substrates and the enzyme. To

optimize the kinetic properties of the substrates,two different

thiol groups,2-mercaptoethylamine- and 4-aminothiolphe-

nol-conjugated 1,2-bis(2-aminoethoxy)ethane linkers, were

connected to the 3’-position of the cephem nucleus. A simple

deprotection (that is,removal of the diphenyl methyl ester at

the 4’-position by trifluoroacetic acid and anisole) followed by

[2]

wide,in both hospital and community settings. Therefore,

detecting Blas and screening their inhibitors in biological

samples before conducting the efficient antibiotic therapy is

extremely important clinically. Some commonly used fluo-

rescent (for example,genotyping based on the polymerase

chain reaction (PCR)) or chromogenic assays (such as

nitrocefin or pyridine-2-azo-p-dimethylaniline cephalosporin

(PADAC) indicators) are currently used successfully to

perform such detections.^[3]Some other fluorogenic substrates

and biocompatible hydrogels have also been developed as

reporters for imaging the gene expression of Blas in vitro and

in vivo.^[4]A simple,rapid,and economical assay is still highly

desirable because current assays have some major drawbacks,

such as laborious manipulation,time-consuming processes,

high reliance on specific instrumentation,and limited chem-

ical stability and aqueous solubility of the individual sub-

strates.

Gold nanoparticles (Au-NPs) have interesting optical and

electronic properties that have served as a versatile platform

for exploring many facets of basic science. Au-NPs can exhibit

plasmon coupling that may red-shift the resonance wave-

length.^[5]Normally,the exact plasmon resonance band can be

determined by the size,shape,medium,and the relative

distance between the particles.^[6]The different agglomeration

states of Au-NPs can result in distinctive color changes. This

extraordinary optical phenomenon makes Au-NPs ideal

chromogenic probes for reporting molecular recognition

events,^[7]such as making a molecular ruler for colorimetric

detection of DNA hybridization,^[8]carbohydrate sensing,^[9]

[*] R. Liu, R. Liew, J. Zhou, Prof. B. Xing

Division of Chemistry and Biological Chemistry

School of Physical and Mathematical Sciences

Nanyang Technological University, Singapore 637616 (Singapore)

Fax: (+65)6791-1961

E-mail: Bengang@ntu.edu.sg

Homepage: http://www.ntu.edu.sg/home/bengang

[**] This work was supported by SEP (RG139/06) and URC (RG56/06)

grants at the Nanyang Technological University. We thank Prof. P. K.

Wong and Dr. Z. Y. Zhong at ICES for the TEM measurement and Dr.

Edwin K. L. Yeow, Dr. Brendan P. Orner, and the reviewers for helpful

suggestions.

Supporting information for this article is available on the WWW

under http://www.angewandte.org or from the author.

Angew. Chem. Int. Ed. 2007, 46, 8799 –8803

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Scheme 1. Design of the Bla assay based on color changes during enzyme-induced Au-NP aggregation.

HPLC purification produced the substrates 1 and 2 with yields

of 60 and 66.7%,respectively.

the color of the Au-NP suspension alone remained unchanged

with time. When the intact substrates were added,no further

color changes in the Au-NPs demonstrated that both of the b-

lactam substrates were stable under the experimental con-

ditions. In the presence of the Bla-pretreated substrate 1,no

color change was detected within 30 minutes. A clear change

was only observed after a longer incubation time (> 9 h; data

not shown). In contrast,after adding the Bla-pretreated

substrate 2 into the Au-NPs,the color dramatically changed

from pink red to violet blue within seconds. Significant

changes in the UV/Vis absorption were also identified

30 minutes after mixing the enzyme-treated substrate 2 with

the Au-NPs (Figure 1b). Both a decreased absorbance of the

plasmon band at 520 nm and an increased absorbance at

650 nm were observed with increasing time. The shifting of

the absorbance to a longer wavelength over time correlated

with the color change seen in Figure 1a.

After obtaining the precursors,we tested the ability of Bla

activity to induce the aggregation of Au-NPs. Substrates

(8 mm) were initially incubated with Bla (5 nm) in phosphate-

buffered saline (PBS) buffer solution (pH 7.4) for 20 minutes.

Then,the resulting solutions were transferred into Au-NP

suspensions (15 nm in size and with a concentration of 2.6 nm

as determined by the reported method).^[13]To stabilize the

nanoparticles and also to prevent the interaction of proteins

with gold particles,0.1% poly(ethylene glycol) (PEG) 8000

was added to the reaction mixture.^[12a]As shown in Figure 1a,

The Bla-induced Au-NP aggregates were used to monitor

the process of the enzymatic hydrolysis reactions. Figure 2a

shows the time course for the color change of the gold

particles upon the addition of Bla-pretreated substrates. For

substrate 1,the absence of a significant color change within

30 minutes demonstrates the slow process of the overall

enzyme assay. For substrate 2,about two thirds of the change

in the total absorbance occurred within the first five minutes,

thereby confirming that this reaction process was very fast.

We hypothesized that the colorimetric assay mainly consisted

of a two-step interaction: one was the enzymatic hydrolysis to

release the dithiol linkers and the other was the binding of

cleaved dithiol linkers to the surface of Au-NPs to form an

aggregation. Figure 2b shows the change in the absorbance

recorded for the binding of free dithiol linkers to the Au-NPs

at different times (linker 1: di-2-mercaptoethylamine-conju-

gated 1,2-bis(2-aminoethoxy)ethane and linker 2: di-4-amino-

thiolphenol-conjugated 1,2-bis(2-aminoethoxy)ethane). Both

of the linkers exhibited similar affinities to that of the Au-

NPs,and their rapid binding process,which occurred within

seconds,suggested that the kinetics of the system was limited

to an enzymatic hydrolysis reaction. Therefore,the colori-

metric feature of Au-NPs was applied to measure the

Figure 1. Colorimetric assay of Bla. a) Colors of the Au-NP solution in

the absence or presence of Bla-treated substrates; 1: Au-NPs only; 2:

Au-NPs and substrate 2; 3: Au-NPs and Bla-treated substrate 2; 4: Au-

NPs and substrate 1; 5: Au-NPs and Bla-treated substrate 1. b) UV/Vis

spectra of Au-NPs taken at 2-min intervals for 30 min after the

addition of Bla-treated (5 nm) substrate 2 (8 mm).

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Figure 3. Absorbance change at 650 nm of Au-NPs at 2 h after mixing

60pm Bla-pretreated substrate 2 (8 mm) with various concentrations of

gold particles (ranging from 0.65, 1.3, 2.2, 2.6, 3.0, 3.4, 4.0, 4.8, to

10.4 nm; analyses were performed in triplicate).

one commonly used Bla inhibitor,sulbactam,was selected

and the effect of the enzyme inhibition was evaluated by using

our system. After incubating the inhibitor-pretreated Bla with

substrate 2 and then mixing with Au-NPs,a different color

was observed,which indicated the different properties of the

enzyme inhibition. The IC₅₀value was found to be about

4.4 mm,which was similar to the previously observed value

(Figure 4).^[15]This finding supports our hypothesis that the

color changes can be used for a quantitative analysis of Bla

activity and for screening the Bla inhibitors.

Figure 2. Aggregation kinetics of Au-NPs for Bla. a) Absorbance

change (at 650 nm) of Au-NP suspension with time in the presence of

Bla-pretreated substrates (5 mm): substrate 1 ( ), substrate 2 ( ).

b) Dependence of absorbance change on time for the interactions of

free dithiol linkers (5 mm) with the Au-NP suspension: linker 1 ( ),

linker 2 ( ).

hydrolysis kinetics of Blas. Further analysis of the enzymatic

kinetics of the two substrates at pH 7.4 allowed the determi-

nation of k_cat= (0.33 Æ 0.1) s^À1and K_M= (140 Æ 11) mm for

substrate 1 and k_cat= (8.69 Æ 1.3) s^À1, K_M= (113 Æ 8.0) mm for

substrate 2 (see the Supporting Information). The small value

of k_cat.for substrate 1 demonstrated that the leaving group

based on the alkyl thiol group was less efficient than that of

thiolphenol which was used in substrate 2. This result suggests

that the alkyl thiol based substrate 1 was itself kinetically inert

towards Bla and thus exhibited a slow response to Au-NP

aggregation. A concentration as low as 60 pm of Bla is readily

detectable with substrate 2 and Au-NPs (see the Supporting

Figure 4. Inhibition assay of Bla activity by sulbactam. The reaction

was monitored by the absorbance change at 650 nm (analyses were

performed in triplicate).

Information),which is more sensitive than that of the

[14]

commonly used Bla indicator,nitrocefin.

This detection

Furthermore,the different aggregation behaviors of

substrate 2 with Bla were monitored by TEM. As shown in

Figure 5a,substrate 2 itself (8 mm) was unable to induce the

aggregation of the Au-NP suspensions. Upon treatment of the

same concentration of substrate 2 with Bla (5 nm),the enzyme

interaction triggered the release of the modified dithiol linker,

thus inducing the cross-linking of the Au-NPs and increasing

the aggregation dramatically (Figure 5b). Although some

agglomerations could also be detected in Figure 5a,possibly

caused by self-assembly during the drying process in the

sample preparation,most of the Au-NPs were dispersed

randomly in the solution with diameters of approximately

15 nm. Dynamic light scattering (DLS) measurements further

sensitivity can be profoundly influenced by the concentrations

of Au-NPs. Figure 3 shows the quantitative relationship

between the absorbance change ((AÀA₀)/A₀) at 650 nm and

the different concentrations of gold particles towards 60 pm

Bla. It is clear that the significant change in the absorbance at

the Au-NP concentrations ranging from 1.0 to 4.0 nm results

in a good sensitivity for the colorimetric enzyme assay for Bla.

The largest change in absorbance from dispersed to fully

aggregated nanoparticles was observed when the concentra-

tion of Au-NPs was approximately 2.6 nm.

We also determined the Bla inhibition by using this simple

and sensitive Au-NP assay. To demonstrate this application,

Angew. Chem. Int. Ed. 2007, 46, 8799 –8803

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different types of Blas exhibited different enzymatic con-

versions for the same substrate,which is consistent with

results reported recently.^[4d]A CC1 fluorescent assay was

performed to determine Bla activity in these bacterial strains

(see the Supporting Information).^[4b]As shown in Figure 6b,

no significant fluorescent signal was detected in wild-type E.

coli Bl21. Emission of CC1 in E.coli Bl21 (with Bla) was

approximately four times higher than that in K. pneumoniae,

which confirmed the highest enzyme activity in the Bla-

(TEM-1)-encoded E.coli Bl21 strains. As a commonly used

fluorogenic probe,CC1 was more sensitive in the detection of

Blas than was the colorimetric assay. However,CC1 itself was

not as stable and spontaneous hydrolysis easily occurred. In

addition,the whole fluorescent assay had to be conducted

with specific instrumentation,such as with a fluorometer or a

fluorescent microscope.

Figure 5. TEMs of a) substrate 2 (8 mm) in Au-NPs only and b) incuba-

tion of substrate 2 (8 mm) with Bla (5 nm) in Au-NP solutions. Scale

bars: 200 nm.

confirmed a well-dispersed population of Au-NPs in the

solution with substrate 2 only and highly aggregated Au-NPs

in the solution with Bla-pretreated substrate 2 (see the

Supporting Information).

To evaluate whether substrate 2 would respond to Bla in

living bacterial cells,different strains of bacteria (ca.

10⁸cfumL^À1; cfu = colony-forming units) such as wild-type

E. coli Bl21,antibiotic-resistant plasmid-encoded E.coli Bl21,

and one clinically isolated b-lactam-resistant K. pneumoniae

(ATCC 700603) were incubated with substrate 2. As shown in

Figure 6a,substrate 2 treated with wild-type E. coli Bl21 do

The colorimetric assay was also performed using nitro-

cefin,a standard Bla indicator (see the Supporting

Information). The result indicated that nitrocefin could

induce a similar color change from yellow to pink in both of

the b-lactam-resistant bacteria. There was no color difference

between the Bla-encoded E.coli Bl21 and the K. pneumoniae

strains. Moreover,the pink color was also detected in the

wild-type E. coli Bl21 bacteria where no Bla was present,

which is possibly because of the nonspecific hydrolysis of

nitrocefin. Therefore,compared with the nitrocefin-based

colorimetric assay,the significantly different color change

observed in different b-lactam-resistant bac-

teria and the absence of background activity in

wild-type bacterial strains in the Au-NP-based

colorimetric assay indicate that the latter has a

higher reporting threshold than that of the

nitrocefin assay. This finding suggests that the

Au-NP-based enzymatic assay provides a par-

ticularly useful reporting approach for systems

that have significant background activity,

which would cause a false-positive signal in

the nitrocefin assay. This new Au-NP-based

colorimetric assay induced by an enzymatic

reaction leads to an alternative approach for

the simple and specific indication of living

drug-resistant bacteria in real time.

Figure 6. a) Colorimetric assay of the Au-NP aggregation at 30 min after incubation with

bacterial strains; 1: Au-NPs only; 2: substrate 2 (8 mm) treated with wild-type Bl21. 3:

substrate 2 treated with plasmid-encoded Bl21; 4: substrate 2 treated with K. pneumo-

niae. b) Fluorescent emission (I_f) of CC1 (2 mm) in wild-type E. coli Bl21 (green), K.

pneumoniae (black), and plasmid-encoded E. coli Bl21 (red). The excitation was measured

at 360 nm.

In summary,the work presented herein

describes a simple,economical assay to rapidly

visualize Bla activities by using gold nano-

particles. This assay,which involves the use of

Bla to cleave the modified dithiol linker from

the cephem nucleus and to induce the cross-

linking of Au-NPs,does not require specific

not lead to the Au-NPs color change because it is unable to

express Bla. However,significant color change was observed

within 30 minutes after adding plasmid-encoded E. coli Bl21

or substrate 2 treated with K. pneumoniae to the Au-NP

suspensions. The different color changes (violet blue in Bla-

encoded E.coli Bl21 and reddish purple in K. pneumoniae)

were attributed to the different Bla expressions. These two b-

lactam-resistant bacterial cells contained different kinds of

extended-spectrum b-lactamases (ESBLs; TEM-1 in plasmid-

encoded E. coli Bl21 and SHV-18 in K. pneumoniae).^[16]The

instrumentation or complicated experimental steps. It can

offer an alternative platform to evaluate the enzymatic kinetic

reactions and to screen Bla inhibitors in real time. It may also

provide useful practical applications for the rapid and specific

detection of antibiotic-resistant bacteria in clinical settings.

Development of a new type of the Au-NP-based colorimetric

b-lactam substrates with small molecular weights,efficient

cell permeability,and fast pharmacokinetic properties is

currently ongoing.

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deionized water and Bla was dissolved in PBS buffer solution

(pH 7.4) to make different Bla concentrations. Then,Bla solution

(10 mL) was mixed with the substrates (190 mL) for the enzyme

interactions. The Bla-pretreated substrates were transferred into a

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Received: June 23,2007

Revised: August 22,2007

Published online: October 17,2007

Keywords: b-lactamase · bacterial cells · colorimetric assays ·

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