Sensitive, Simultaneous Quantitation of Two Unlabeled DNA Targets Using a Magnetic Nanoparticle–Enzyme Sandwich Assay

We report herein the development of a simple, sensitive colorimetric magnetic nanoparticle (MNP)–enzyme-based DNA sandwich assay that is suitable for simultaneous, label-free quantitation of two DNA targets down to 50 fM level. It can also effectively discriminate single-nucleotide polymorphisms (SNPs) in genes associated with human cancers (KRAS codon 12/13 SNPs). This assay uses a pair of specific DNA probes, one being covalently conjugated to an MNP for target capture and the other being linked to an enzyme for signal amplification, to sandwich a DNA target, allowing for convenient magnetic separation and subsequent efficient enzymatic signal amplification for high sensitivity. Careful optimization of the MNP surfaces and assay conditions greatly reduced the background, allowing for sensitive, specific detection of as little as 5 amol (50 fM in 100 μL) of target DNA. Moreover, this sensor is robust, it can effectively discriminate cancer-specific SNPs against the wild-type noncancer target, and it works efficiently in 10% human serum. Furthermore, this sensor can simultaneously quantitate two different DNA targets by using two pairs of unique capture- and signal-DNA probes specific for each target. This general, simple, and sensitive DNA sensor appears to be well-suited for a wide range of genetics-based biosensing and diagnostic applications.

= 7.2) at room temperature for 1 h, leading to the MNP surface being functionalised with maleimide groups. Thereafter, the MNPs were washed by Buffer C twice, and then 5 nmol of thiolated capture-DNA in 1 mL Buffer C was added into the above MNPs and incubated for 1 h at room temperature. The capture-DNA is covalently conjugated to the MNP surface via Michael addition of the thiolate to the MNP surface maleimide groups (see Scheme S1). The MNPs were subsequently washed twice by Buffer C. It should be noted that all of the original and washing supernatants were collected and combined for UV measurement at 260 nm to determine the amount of free-unbound capture-DNA, allowing the estimation of the capture-DNA conjugation efficiency on the MNP. The capture-DNA loaded MNPs were then treated with 5 μL of 2-mercaptoethanol in 1 mL Buffer A (PBS plus 1 mg/mL BSA) to cap any unreacted maleimide groups and to block the MNP surface to reduce non-specific absorption of HRP-NAV. Typically, the capture-DNA surface loading on the MNP is around 0.5 nmol/mg of MNP.

B) General procedures for DNA detection using HRP based assay
The capture DNA (cDNA) was covalently conjugated to an amine modified MNP surface via a PEG containing hetero-functional cross linker SM(PEG) 12 as described in the SI. The PEGylated cross-linker is used here because of its well-know ability of resisting non-specific adsorption [17][18][19] , allowing greatly reduced assay background and hence achieving higher sensitivity. The amount of cDNA loaded on the MNP was estimated by using our previously established method. 19 The signal DNA (sDNA) was linked to neutravidin-(NAV-) HRP or ALP conjugate via the strong, specific biotin-NAV interaction at 1 to 1 molar ratio. NAV-En conjugate was selected here because it can offer significantly reduced non-specific adsorption compared to the avidin-or streptavidin-enzyme conjugate. A certain amount of MNP-cDNA was mixed with 5 pmol HRP-sDNA and various amounts of target DNA. The final volume of each sample is 500 μL in PBS buffer (containing 1 mg/mL BSA). After 1 h incubation at room temperature, all samples were washed by PBS buffer once, by PBS buffer (containing 0.1% Tween-20) twice, by PBS buffer once again to remove any unbound target DNA and HRP-sDNA. Then the MNP sandwiches were dispersed in 380 μL PBS, the enzymatic amplification was initiated by the addition of 50 μL amplex red (0.2 mM) and 50 μL H 2 O 2 (0.2 mM). After a fixed period for enzymatic amplification, 20 μL N 3 Na (1 M) was added into each sample to terminate the enzymatic assay and the UV-vis absorption spectra of all supernatants were recorded.
For SNP discrimination of experiment, the detection procedures were effectively same as those described above. Specifically, MNP-cDNA3 (20 μg) and 5 pmol HRP-sDNA3 are mixed with the full complementary T-DNA3, or their SNP targets (T-DNA4 or T-DNA5) at identical concentrations (100 pM) in 500 μL PBS buffer, and after incubation and washing procedures, only 10 min of enzymatic amplification time was conducted.

C) Limit of detection challenges
To evaluate the limit of detection of this proposed sensor using colorimetric readout, the assay procedures were basically the same as those described above, except where 10 g MNP-cDNA3 and 0.8 pmol HRP-sDNA3 were used to detect different amounts of T-DNA3 in 200 μL PBS buffer. After incubation and washing procedure, resulting MNP-dsDNA-En assemblies were dispersed in 200 μL PBS buffer containing amplex red and H 2 O 2 (5 μM each). Subsequently, real-time fluorescence change was monitored on an Envision plate reader using BODIPY TMR FP 531 as excitation filter and Cy3 595 as emission filter.

D) General procedures for DNA detection using ALP
For single DNA detection using ALP as signal amplifier, MNP-cDNA2 (20 μg) were mixed with 5 pmol ALP-sDNA2 and T-DNA2 of various concentrations. The final volume of each sample is 500 μL in Tris buffer (containing 1 mg/mL BSA). Because the phosphate could inhibit the enzymatic ability of ALP, 20 Tris buffer was employed instead of PBS buffer in preparing samples and washing procedures. Then the MNP sandwiches were dispersed in 430 μL Tris buffer, and then each was mixed with 50 μL FDP (0.2 mM) initiating the enzymatic amplification assay. After 1 h, following the addition of 20 μL PBS buffer to stop the enzymatic reaction, the UV spectra of supernatants of all the samples were recorded.

E) Simultaneous quantitation of two different DNA targets
MNPs-cDNA1 and MNP-cDNA2 (15 μg each) were mixed with 15 μL ALP-sDNA1 (0.25 μM) and 15 μL HRP-sDNA2 (0.25 μM) to quantitate two different target DNA strands, T-DNA1 and T-DNA2. All assays and washing steps were carried out in Tris buffer, because PBS can efficiently inhibit the ALP activity. After sandwich formation and washing, the resulting MNP sandwiches were dispersed in 330 μL Tris buffer, and then each sample was added a mixture of 50 μL FDP (0.2 mM), 50 μL Amplex red (0.2 mM) and 50 μL H 2 O 2 (0.2 mM) to initiate the enzymatic amplifications. After 1 h, 20 μL PBS mixed with 20 μL N 3 Na (1 M) were added simultaneously to stop both enzymatic reactions, the resulting UV spectra of supernatants were then recorded.       Table S1. Comparison of the sensing performances of the MNP-enzyme sandwich assay against some recently reported DNA assays. (No. = number of target detected; LOD = limit of detection; S/N = signal to noise ratio)