#104

Flow-Injection SPR: Sensitive Determinations of Heavy Metal Ions
Flow-injection SPR has been demonstrated as a viable alternative for sensitive detection of heavy
metal ions at trace levels [1-4]. The impetus behind using SPR for elemental analysis stems from its
high sensitivity, simplicity, compact design (for possible field-based work), and universal detection
mechanism (e.g., any elemental species adsorbed onto the SPR sensor can cause a detectablesignal). The challenge, however, is to prepare a chemically or biologically modified sensor chip that isspecific to a given analyte without interferences from other species present in the sample solution. Zareand coworkers have showed that an alkanedithiol-covered gold surface is selective to mercury ion(Hg2+) [1], and Forzani et al. attached oligopeptides onto gold films for selective determinations of Ni2+
and Cu2+ [2]. Zhang et al. recently constructed a sensor surface covered with apo-metallothionein (i.e.,metal-free metallothionein, a cysteine-rich protein) for selective determinations of Hg2+ and Cd2+ [3].
The principle behind this detection mechanism is illustrated in FIG. 1. As metal ions are complexed by
the apo-metallothionein (apo-MT) molecules, the resultant protein conformational changes can besensitively measured by SPR. The extent of the conformational changes (or thickness variation of theimmobilized proteins) is proportional to the amount of metal ions in the sample solution. This method
yielded detection levels for Cd2+ and Hg2+ at 0.1 µM and 5 µM, respectively [3].

(a)

(b)

apo-metallothionein

Metal ion

Metal ionsAuAu

FIG. 1 Schematic representation of the conformation change of apo-MT upon metal binding. The black
dots in (b) represent the metal ions. Notice that the predominant protein attachment is the linkage of MT
molecules via their N-termini.

The two sensorgrams depicted in FIG. 2 suggest that the apo-MT molecules have a stronger
binding affinity with Hg2+ than with Cd2+. When the Cd2+ injection was followed by the introduction ofHg2+ into the flow cell, the Hg2+ signal was much greater than the Cd2+ signal, even though both metal
ions were of the same concentration. If the injection order was reversed (inset of FIG 2), the injection ofCd2+ into the cell housing a MT sensor chip that had already been exposed to Hg2+ did not yieldappreciable SPR dip shifts.
A BI-SPR 1000 system, equipped with a dual-channel flow cell and a PDMS (polydimethylsiloxane)
gasket, was also used for the detection of Hg2+ at a hexanedithiol-modified gold sensor chip. A 10-mM
acetate buffer (pH 4.5) was used as the running buffer and a flow rate of 50 µL/min was used. Mercury
standards of various concentrations were analyzed by injecting 20 µL of sample into the flow cell. FIG.

3a overlaid two representative sensorgrams that show the complexation of Hg2+ at two different
concentrations by the surface-bound hexanedithiol. The calibration curve (FIG. 3b) exhibited excellent
linearity between sub-µM to mM (R2 = 0.999). As demonstrated by Zare and coworkers, a carefully
designed protocol based on this approach can lead to a detection level for Hg2+ down to sub-nM [1].
FIG.
2ASPR
sensorgram
2+corresponding to the injection of Hg
into the flow cell housing a gold2+
sensor chip covered by Cd 2+
reconsitituted MT. 50 µM Cd and2+50µMHg
wereinjectedsequentially at a flow rate of 1 mL/h.
Inset: The apo-MT was first2+
reconstituted with Hg by injecting
2+
100 µM Hg . This was followed by2+
injecting 100 µM Cd .

0.6

(a)

SPR Angle (Degree)

SPR angle (mdeg.)

60

4020004080

120

Time (Sec.)

(b)
0.4

0.2

0.0
0.0

0.5

1.0

1.5

2.02+

[Hg ] (mM)

FIG. 3 (a) Time-resolved SPR signals corresponding to the injections of 10 µM (black curve) and 2.5 µM
2+(red curve) Hg into a flow cell housing a hexanedithiol-covered gold sensor chip. (b) Calibration curve
2+constructed by plotting the SPR response as a function of Hg concentration.

References[1] Chah, S.; Yi, J.; Zare, R. N. Sens. Actuators B : Chem. 2004, 99 : 216-222.
[2] Forzani, E. S.; Zhang, H.; Chen, W.; Tao, N. Environ. Sci. Technol. 2005, 39:1257-1262.
[3] Zhang, Y.; Xu, M.; Wang, Y.; Toledo, F.; Zhou, F. Sens. Actuators B. 2007, 123, 784-792.
[4] Wu, C.-M.; Lin, L.-Y. Biosens. Bioelectron. 2004, 20:864-871.

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