Our detection is based on the fact that 1) anti E. coli antibody-conjugated nanorods can readily and specifically identify Escherichia coli O157:H7 bacterium, through antibody–antigen recognition (as shown in ) and ) when anti E. coli antibody-conjugated nanorods (as shown in ) were mixed with various concentrations of Escherichia coli O157:H7 bacterium, two-photon scattering intensity increases by about 40 times. This increment is due to the fact that since E. coli bacteria is more than an order of magnitude larger in size (1–3 micro meter (µm)) than the anti E. coli antibody-conjugated gold nanorods. In the presence of E. coli bacteria, several gold nanorods conjugates with one E. coli bacteria and as a result anti E. coli antibody-conjugated gold nanorods undergo aggregation (as shown in ). Due to the aggregation, a new broad band appears around 200 nm far from their longitudinal plasmon absorption band and color change takes place ( as shown in ). This bioassay is rapid, takes less than 15 min from bacterium binding to detection and analysis, convenient, and highly selective.
Figure 1 A) Schematic representation of anti E. coli antibody-conjugated nanorods based sensing of E. coli bacteria. B) TEM image of anti E. coli antibody-conjugated nanorods before addition of E. coli bacteria, C) Photograph showing colorimetric change upon addition (more ...)
Figure 2 A: Plot demonstrating two-photon scattering intensity changes (by 40 times) due to the addition of E. coli bacteria to anti E. coli antibody conjugated gold nanorods. Two-photon scattering intensity changes very little upon addition of Salmonella bacteria. (more ...)
demonstrates how the two-photon scattering intensity varies due to the addition of E. coli bacteria to anti E. coli antibody conjugated gold nanorods. We observed a very distinct two-photon scattering intensity change (4 times) after the addition of 50 cfu/ml E. coli bacteria. After the addition of E. coli bacteria to anti E. coli antibody conjugated gold nanorods, the two-photon Rayleigh scattering (TPRS) intensity change observed in our assay, can be due to several factors. 1) The intensity of two-photon scattering signal from gold nanorod solution can be expressed as 14–16,20,34–42
is a geometric factor, Nw
the number of water molecules and gold nanorods per unit volume, βω
are the quadratic hyperpolarizabilities of a single water molecule and a single gold nanoparticle, ε2ω
is the molar extinction coefficient of the gold nanoparticle at 2ω, l is the path length and Iω
the fundamental intensity. The exponential factor accounts for the losses through absorption at the harmonic frequency. Since there is a center of inversion in nanorod, the TPRS intensity arising from gold nanorods cannot be due to electric dipole contribution. Considering the size of a nanorod, the approximation that assumes that the electromagnetic fields are spatially constant over the volume of the particle may not suitable anymore. As a result, the total nonlinear polarization consists of different contributions such as multipolar radiation of the harmonic energy of the excited dipole and possibly of higher multipoles, as we discussed in our previous publication or reported by others. 14–16,20,34–42
The HRS intensity therefore also consists of several contributions. The first one is the electric dipole approximation, which may arise due to the defects in nanoparticle. This contribution is actually identical to the one observed for any non-centrosymmetrical point-like objects such as efficient rod-like push-pull molecules. The second contribution is multipolar contribution like electric quadrupole contribution. This contribution is very important when the size of the particle is no longer negligible when compared to the wavelength, as we reported before. Since E. coli bacteria is more than an order of magnitude larger in size (1–3 micrometer (µm)) than the anti E. coli antibody-conjugated gold nanorods, several gold nanorods conjugates to one E. coli
bacteria and as a result, anti E. coli
antibody-conjugated gold nanorods undergo aggregation in the presence of E. coli
bacteria (as shown in ). Due to the aggregation in the presence of E. coli
bacteria, nanorods looses the center of symmetry and as a result, one can expect significant amount of electric dipole contribution to the two-photon scattering intensity. Since electric dipole contributes several times higher than that of multipolar moments, we expect two-photon scattering intensity to increase with aggregation. 2) When E. coli
bacteria is added to the anti E. coli
antibody conjugated gold nanorods, a clear colorimetric change is observed due to the aggregation. As shown in , absorption maximum for longitudinal absorption band at 680 decreases with the increase of the concentration of E. coli
bacteria, whereas a new broad band corresponding to the absorption of nanorod aggregates at 950 nm increases with the increment of the concentration of E. coli
bacteria. According to the two-state model, 43
where ω is the fundamental energy of the incident light, µeg
is the transition dipole moment and ωeg
is the transition energy between the ground state |g> and the charge-transfer excited state |e>, Δµeg
is the difference in dipole moment between |e> and |g> states.
, and λmax
shifted 270 nm towars red upon addition of bacteria (as shown in ), β should change tremendously upon the addition of bacteria and as a result the two-photon scattering intensity should changes tremendously with the addition of E. coli
bacteria. 3) The single photon resonance enhancement as well as two-photon luminescence factors are much larger for nanorod aggregates due to the closeness of λmax
to the fundamental wavelength at 860 nm. This factors should increase two-photon scattering intensity. (4) Since size increases tremendously with aggregation, the two-photon scattering intensity should increase with the increase in particle size. 5) The aggregation of gold nanorods can enhance the scattering intensity because the local electric field enhancement becomes larger owing to the surface plasmon resonance coupling.
We also noted that though two-photon scattering intensity changes about 4 times even at the concentration of 50 Colony Forming Units/ml (cfu/ml) of E. coli bacteria, the visible color changes can be observed only after the addition of 10,000 cfu/mL bacteria, which indicates that our two-photon scattering based gold nanorod assay is about 2 orders of magnitude more sensitive that the usual colorimetric technique.
To understand whether our assay is highly selective, we have also performed how two-photon scattering intensity changes upon addition of Salmonella typhimurium bacteria to anti E. coli antibody conjugated gold nanorods. As shown in , two-photon scattering intensity changes only 6% when we added the Salmonella typhimurium bacteria to anti E. coli antibody conjugated gold nanorods. Similarly when we added E. Coli bacteria to anti salmonella antibody conjugated gold nanorods, two-photon scattering intensity changes only 5 %. So the above data demonstrate that our assay is highly selective. To evaluate whether the our assay is selective to O157:H7 in the presence of other E. coli strains, we have also measured two-photon scattering intensity changes upon addition of different E. coli strains O157:H7, O157:NM and O157:non-H7 separately . As shown in , two-photon scattering intensity changes only 1.7–2.4 times when we added O157:NM and O157:non-H7 strains to monoclonal antibodies (MAbs 2B7) coated gold nanorod colloidal solution. So our results shows that our assay is quite selective over other E.Coli strains.
Figure 3 Plot demonstrating selectivity of our two-photon scattering assay over different E.Coli strains. Two-photon scattering intensity changes 40 times due to the addiction of E.Coli O157:H7 strains to specific anti E. coli antibody (MAbs 2B7) conjugated gold (more ...)
Our results also indicate that (as shown in ), the TPRS intensity changes linearly with the concentration of the E. coli bacteria at the lower concentration range. To evaluate whether our assay is capable of measuring E. coli bacteria concentration quantitatively, we performed two-photon Rayleigh scattering intensity measurements at different concentrations of target E. coli bacteria at lower concentration range. As shown in , the two-photon scattering intensity increment is highly sensitive to the concentration of target E. coli bacteria over the range of 50–2100 cfu/ml and the intensity increased linearly with concentration. Our data indicate that our assay exhibits detection limit to detect E. coli bacteria as low as 50 cfu/ml. So our gold nanorod based two-photon scattering assay can provide a quantitative measurement of E. coli bacteria concentration over 50–2100 cfu/ml concentration range.
Plot demonstrating linear correlation between two-photon scattering intensity and concentration of E. coli bacteria over the range of 50–2100 cfu/ml with R =0.985