Figure 1 shows the scheme of a LFA; it consists of three to four different parts:
sample pad, where the liquid sample is applied; conjugate pad (CP) which possesses
the dried AuNP conjugates; membrane (mostly nitrocellulose) which has one test
and one control zone; and absorbent pad (AP), which soaks up the test liquid. The
LFA uses the capillary force; therefore the sample flows through all the pads while
collecting the AuNPs of the CP, interacting on the membrane and finally reaching
the AP [35, 38].
The analyte concentration in the sample can affect the assay and result in a
concentration-dependent intensity of the test zone, thereby allowing quantification.
For reliable results LFAs have a readout window after running the test; afterward the
signals can change due to detachment of the particles or analytes. In general they are
point-of-care devices that deliver a fast result and are easy to use, even for untrained
personnel [38] and exist for various medical targets, as shown in Table 1. The listed
LFAs are currently still restricted to research applications, but the usage in complex
samples for diagnostic analysis is assumed to allow clinical use.
2.1.2 Colorimetric Assays
In contrast to LFAs, which use a membrane as a solid phase, AuNPs can also be used
in liquid-phase assays. Colorimetric assays with gold nanoparticles use the color
shifting effect from red to purple/blue, when AuNPs aggregate or assemble
[39]. There are two different setups, one with aptamers adsorbed on the particles
and the other with stable aptamer-AuNP conjugates.
Colloidal AuNPs have a special localized surface plasmon resonance (LSPR),
resulting in their optical properties. The LSPR is sensitive to the size of the AuNPs
and the local refractive index near their surface. Changes in the LSPR are visible due
to the color change of the AuNPs [40]. AuNPs synthesized with citrate are stable in
solution because the citrate ions form an adsorbed protective layer around the
AuNPs, and the electrostatic repulsion from these anions keeps the AuNPs separated
[41]. When salt (e.g., NaCl) is added, the AuNPs aggregate, because the salt ions
shield the negative charge on the AuNPs and the interparticle distance decreases,
thereby the LSPR changes and the AuNPs solution turns purple [42, 43].
Ligands like aptamers can be bound to the AuNPs and stabilize the AuNPs at
higher salt concentrations [44]. Aptamers can form electrostatic interactions with
AuNPs and adsorb reversibly on their surface or they can be conjugated covalently
or covalently like, as described in Sect. 1.1. The negatively charged aptamers
provide stabilization of AuNPs by electrostatic repulsion and steric stabilization,
so that the AuNP solution retains it red color. If an analyte is added and two aptamers
bind in a sandwich setup simultaneously, the AuNPs assemble and the interparticle
distance gets reduced; therefore the LSPR shifts and the visible color changes to
purple [39].
The different colorimetric assay setups are shown in Fig. 2.
Salt-induced aggregation assays with adsorbed aptamers are shown in Fig. 2a,
and directed assembly of conjugates is shown in Fig. 2b.
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A. Eilers et al.
sample pad, where the liquid sample is applied; conjugate pad (CP) which possesses
the dried AuNP conjugates; membrane (mostly nitrocellulose) which has one test
and one control zone; and absorbent pad (AP), which soaks up the test liquid. The
LFA uses the capillary force; therefore the sample flows through all the pads while
collecting the AuNPs of the CP, interacting on the membrane and finally reaching
the AP [35, 38].
The analyte concentration in the sample can affect the assay and result in a
concentration-dependent intensity of the test zone, thereby allowing quantification.
For reliable results LFAs have a readout window after running the test; afterward the
signals can change due to detachment of the particles or analytes. In general they are
point-of-care devices that deliver a fast result and are easy to use, even for untrained
personnel [38] and exist for various medical targets, as shown in Table 1. The listed
LFAs are currently still restricted to research applications, but the usage in complex
samples for diagnostic analysis is assumed to allow clinical use.
2.1.2 Colorimetric Assays
In contrast to LFAs, which use a membrane as a solid phase, AuNPs can also be used
in liquid-phase assays. Colorimetric assays with gold nanoparticles use the color
shifting effect from red to purple/blue, when AuNPs aggregate or assemble
[39]. There are two different setups, one with aptamers adsorbed on the particles
and the other with stable aptamer-AuNP conjugates.
Colloidal AuNPs have a special localized surface plasmon resonance (LSPR),
resulting in their optical properties. The LSPR is sensitive to the size of the AuNPs
and the local refractive index near their surface. Changes in the LSPR are visible due
to the color change of the AuNPs [40]. AuNPs synthesized with citrate are stable in
solution because the citrate ions form an adsorbed protective layer around the
AuNPs, and the electrostatic repulsion from these anions keeps the AuNPs separated
[41]. When salt (e.g., NaCl) is added, the AuNPs aggregate, because the salt ions
shield the negative charge on the AuNPs and the interparticle distance decreases,
thereby the LSPR changes and the AuNPs solution turns purple [42, 43].
Ligands like aptamers can be bound to the AuNPs and stabilize the AuNPs at
higher salt concentrations [44]. Aptamers can form electrostatic interactions with
AuNPs and adsorb reversibly on their surface or they can be conjugated covalently
or covalently like, as described in Sect. 1.1. The negatively charged aptamers
provide stabilization of AuNPs by electrostatic repulsion and steric stabilization,
so that the AuNP solution retains it red color. If an analyte is added and two aptamers
bind in a sandwich setup simultaneously, the AuNPs assemble and the interparticle
distance gets reduced; therefore the LSPR shifts and the visible color changes to
purple [39].
The different colorimetric assay setups are shown in Fig. 2.
Salt-induced aggregation assays with adsorbed aptamers are shown in Fig. 2a,
and directed assembly of conjugates is shown in Fig. 2b.
166
A. Eilers et al.